DNA-RNA hybrids arise in all cell types, and are removed by multiple enzymes, including the trimeric ribonuclease, RNase H2. Mutations in human RNase H2 result in Aicardi–Goutières syndrome (AGS), an inflammatory brain disorder notable for being a Mendelian mimic of congenital viral infection. Previous studies have shown that several AGS-associated mutations of the RNase H2B subunit do not affect trimer stability or catalytic activity and are clustered on the surface of the complex, leading us to speculate that these mutations might impair important interactions of RNase H2 with so far unidentified proteins. In this study, we show that AGS mutations in this cluster impair the interaction of RNase H2 with several members of the CoREST chromatin-silencing complex that include the histone deacetylase HDAC2 and the demethylase KDM1A, the transcriptional regulators RCOR1 and GTFII-I as well as ZMYM3, an MYM-type zinc finger protein. We also show that the interaction is mediated by the zinc finger protein ZMYM3, suggesting that ZMYM3 acts as a novel type of scaffold protein coordinating interactions between deacetylase, demethylase and RNase H type enzymes, raising the question of whether coordination between histone modifications and the degradation of RNA-DNA hybrids may be required to prevent inflammation in humans. Aicardi–Goutières syndrome (AGS) is a rare, largely autosomal-recessive disorder characterised by microcephaly, basal ganglia calcification and elevated levels of lymphocytes and interferon-alpha in the cerebrospinal fluid, with occasional extra-neurological involvement of the liver, spleen and skin ,2. AGS uConsistent with a central role for interferon in the disease, AGS has been associated with heterozygous gain-of-function mutations in the dsRNA sensor Interferon Induced With Helicase C Domain 1 IFIH1 MDA5), which increase its affinity for RNA as well as baseline and ligand-induced interferon signalling . AGS has, which iin vitro ) significantly diminished (but did not abolish) interaction with endogenous RCOR1 and KDM1A, and that either one of these zinc fingers alone (fragments 620–678 or 661–719) was sufficient to bind to RCOR1 and KDM1A , RCOR1 (CoREST) and GTFII-I. As the eighth and ninth zinc fingers of the closely-related ZMYM2 protein have been shown to bind to the complex comprising HDAC1, KDM1A and RCOR1 , we sougnd KDM1A . Using tnd KDM1A .As its name suggests, ZMYM3 is a member of a family of proteins, the six members of which each possess arrays of MYM-type zinc finger motifs . As showin vitro phenotype of targeted Zmym3-/ murine ES cells in vitro The experiments presented here identify RNase H2 as a novel component of the co-repressor of transcription KDM1A/RCOR1/HDAC2 LCH complex, via its interaction with ZMYM proteins ZMYM3 and ZMYM2. ZMYM2 and 3 have been shown to associate with the KDM1A/RCOR1/HDAC2 complex , althougOur results identify several novel interactions of RNase H2, including the uncharacterised zinc finger protein ZMYM3, the transcription factor TFII-I (GTFII-I), the transcription repressor RCOR1, the histone deacetylase HDAC2 and the histone demethylase LSD1 (KDM1A), all of which bind to ZMYM3 . We furt[Δ863–942]) or by C-terminal truncation of the protein  did not lead to major changes in the nuclear distribution  either directly , or vice versa, and that failure to co-ordinate these processes in humans may contribute to the inflammatory brain disorder AGS. The well-established but functionally poorly understood interaction of lncRNAs or small RNAs with several chromatin remodelling and silencing complexes like CoREST or the NuRD and polycomb complexes [By diminishing the interactions of RNase H2B with ZMYM3, it is conceivable that the AGS-mutations investigated here may reduce the stability of the RNase H2 complex, as has been observed for several other AGS-associated RNase H2 mutations that compromise the interaction between the C and A or C and B subunits ,30. Howeomplexes  might exFailure to coordinate the activity of RNase H2 and the CoREST complex could also lead to an alternative source of nucleic acid accumulation and inflammation, by impairing the ability of the CoREST complex to suppress the expression of endogenous transcripts  or by peRegarding the RNase H2/ZMYM3 interaction, our data suggest that other members of the ZMYM family might act redundantly, since we did not observe mayor abnormalities in mouse ES cells devoid of ZMYM3, unlike the differentiation block associated with KDM1A/LSD1 deficiency . Recent N-terminally FLAG-tagged RNase H2B and mutant versions were assembled by PCR using a cDNA template isolated from a human spleen cDNA library (Life Technologies) and using primers introducing an Nhe I restriction site, KOZAK consensus sequence GCCACC, glycine-alanine linker and FLAG (DYKDDDDK)-encoding sequence 5’ of the protein-coding region (primer 2B N-FLAG Fwd), and a Not I restriction site 3’ of the protein-coding region (primer 2B N-FLAG Rev). Nhe I and Not I PCR products and the vector pIRESpuro3 (Clontech) were digested with NheI and NotI, gel purified, ligated to one another to produce pIRESpuro3-2B . pIRESpuro3-2B  and pCDNA4/TO-IRES-GFP, a mammalian expression vector which expresses the gene of interest and EGFP under the control of a CMV promoter (Addgene), were digested with NdeI and NotI, gel purified (the NdeI/NotI fragment containing the RNase H2B gene and the NdeI/NotI fragment containing the backbone of pCDNA4/TO-IRES-GFP) and ligated to produce pCDNA4/TO-2B-IRES-GFP .N-terminally HA-tagged ZMYM3 was generated from a cDNA clone of ZMYM3 Isoform 1  as Afl II/Not I fragments with KOZAK/GA linker/HA tag 5’ of the protein-coding region using primers hsZMYM3 N-HA Fwd/hsZMYM3 Rev and expressed in pCDNA4/TO-ZMYM3-IRES-tdTomato. hsZMYM1,2,4 and 6 were isolated from a human spleen cDNA library (Life technologies), as BamH I/Not I PCR fragments containing the KOZAK/GA/HA module using primers hsZMYM1/2/4/6 N-HA Fwd / hsZMYM1/2/4/6 Rev and introduced into pCDNA4/TO-IRES-tdTomato.ZMYM3 truncation mutants were generated by PCR from pCMV-SPORT6-ZMYM3 as before. All C-terminal truncations (unless otherwise indicated) contained two nuclear localisation signals from the DUF3504 domain of ZMYM3 immediately 3’ of the truncation site while the zinc finger truncation mutants all included the KOZAK/GA/HA module as well as the two predicted nuclear localisation signals from the DUF3504 domain of ZMYM3 and a stop codon. FLAG-tagged ZMYM3 zinc constructs were assembled by PCR as before into pCDNA4/TO-IRES-tdTomato. All primers are listed in -1 penicillin and streptomycin (Sigma) and 10% foetal bovine serum (FBS) (Life Technologies) were transfected with 1 μg of plasmid DNA using GeneJuice Transfection Reagent (Novagen) as per the manufacturer’s instructions. Expression of the fluorescent reporter genes was used to monitor transfection efficiency and ~ 4x106 cells were washed in PBS, harvested and lysed in 20 mM HEPES, 150 mM NaCl, 0.2% Triton X-100 (Sigma), 1X Complete Protease Inhibitor (Roche), 1 mM Dithiothreitol, 2 mM MgCl2 and 3 μl/ml Benzonase (Novagen) at 4°C for 45 mins under gentle rotation. Cell lysates were precleared by centrifugation 19,000 RCF for 20 mins and 460 μl added to pre-washed 15 μl of anti-FLAG M2 affinity gel (Sigma) with30 μl of each lysate set aside to monitor protein input and incubated at 4°C under gentle agitation. Samples were washed 5x in 500 μl PBS supplemented with 0.1% Triton X-100 (Sigma), centrifuged at 100 RCF for 2 mins in Proteus Clarification Mini Spin Columns (Generon) and the bound proteins eluted 2x in 215 μl 3X FLAG peptide at 10 ng/μl (Sigma) and resuspended in 30 μl NuPAGE LDS Sample Buffer (SB) (Life Technologies) supplemented with 50mM DTT.HEK293T cells in Dulbecco's Modified Eagle's Medium (DMEM) supplemented with 100 μg mlFor the immunoprecipitation of endogenous RNase H2B and ZMYM3, 10 μl of Dynabeads Protein A beads (Life Technologies) were twice washed in 1ml of RIPA buffer, and resuspended in 100 μl of RIPA, (with 10 μl of 10 μg/μl purified BSA (NEB)) at 4°C for approximately 45mins, washed three times in 1ml of RIPA, and resuspended in 100 μl of RIPA and then added to 460 μl cell lysates in the presence of 1 μg of rabbit anti-RNase H2B or reagent grade IgG from rabbit serum (Sigma) approximately 0°C for two to three hours. Beads were washed 3x in 1ml RIPA buffer (in 1% Triton X-100) and the pellet resuspended in 30 μl NuPAGE LDS Sample Buffer (SB) (Life Technologies) supplemented with 50mM DTT.Proteins  were separated by SDS-PAGE NuPAGE 4–12% Bis-Tris Gels in 1X NuPAGE MOPS SDS (Life Technologies) and transferred into Immobilin P PVDF membranes (Millipore) in a tris-glycine buffer containing 10% methanol. Membranes were washed 3x5mins in PBS supplemented with 0.05% TWEEN20  (PBST) under gentle agitation, blocked in PBST supplemented with 5% w/v dried skimmed milk (Marvel) for 45mins and washed 3x5mins in PBST prior to incubation with antibody in PBST supplemented with 2.5% (w/v) milk or PBS at 4°C. Dilutions and antibodies used are specified in For protein identification 35 μl of per sample were separated, gels were fixed and stained using the SilverQuest Staining Kit (Life Technologies), or SYPRO Ruby Protein Gel Stain (Life Technologies), according to the manufacturer’s instructions. Bands of interest were excised and identified by tandem LC-MS/MS mass spectrometry, performed by the mass spectrometry facility at the MRC Laboratory of Molecular Biology, UK.Transfected HEK293T cells were grown on poly-l-lysine (Sigma) treated coverslips (Thermo Scientific) for 36 hours and fixed in 1 ml of 4% paraformaldehyde for 20mins at room temperature, washed 3x in 1ml of PBS, permeabilized in 1ml of 0.4% Triton X-100 for 5mins, then washed again in 1ml of PBS 3x 5mins. After blocking in 250 μl of PBS supplemented with 10% normal goat serum (Life Technologies), 0.1% Triton X-100 and 2.5% FBS, cells were stained in 250 μl of blocking buffer for 1hr at room temperature, then washed with 1ml PBS 3x5mins, before being stained with the appropriate dilution see  of seconZmym3tm1(NOCOM)Mfgc) were obtained from the NorCOMM consortia (clone N00012P0_C_1W_B6). The targeting of Zmym3 was confirmed by long range PCR and sequencing and Southern Blotting as described previously [Mouse ES cells (C2-C57BL/6NTac) with a targeted inactivation of Zmym3 Click here for additional data file.S2 Table(PDF)Click here for additional data file.S1 FigProtein extracts from control and HEK293T cells transfected with Flag-tagged RNase H2B were incubated with anti-FLAG beads, and the associated proteins were separated by SDS-PAGE and silver stained to reveal any differences in binding between the wild type protein and mutants corresponding to the single amino acid substitutions indicated. An empty vector (control) transfection was also performed to distinguish specific from non-specific binding. The region indicated by the open arrow indicates the region corresponding to the RNase H2B, specific binding identified by mass spectrometry as ZMYM3.(TIF)Click here for additional data file.S2 FigConfocal micrographs of wild-type and mutant human Flag tagged RNase H2B transfected into HEK293T cells. DAPI stained nuclei and A GFP reporter driven by an IRES was used to monitor transfection and subcellular localisation monitored by staining with M2 anti-Flag antibody and Alexa Fluor 568 Goat anti-Mouse IgG1. A: Wild-type. B: S159I. C: K162T. D: T163I. E: V185G. F: Empty vector control. i: M2 anti-FLAG and. ii: DAPI. iii: GFP expression. iv: Merge. Magnification: 60x.(TIF)Click here for additional data file.S3 Fig(A) Comparison of the proline rich region in ZMYM3 homologues with identical residues shown in red. The numbering corresponds to the amino acids positions in human (NP_005087), mouse (XP_011245953), zebrafish (XP_005159763) and fly (NP_001097946) proteins. (B) Schematic representation of the domain structure of human ZMYM3 including the secondary structure predictions  from Phyre2 . Predict(EPS)Click here for additional data file.S4 FigThe subcellular localization of HA-tagged ZMYM3 and the truncation mutants used to map the biochemical interactions with RNase H2B monitored by confocal microscopy. HEK293T cells stained with Mouse anti-HA antibodies and Alexa Fluor 568 Goat anti-Mouse counterstained with DAPI 24 hours post-transfection. Magnification = 60x. A schematic representation of the full-length protein and the deletion fragments are indicated below the corresponding panels.(TIF)Click here for additional data file.S5 FigZmym3 locus and the NorCOMM targeting strategy. The location of the coding exons is shown as brown boxes and the regions of homology flanking Exon3 used for targeting are shown in blue. Bcl I restriction sites are shown for guidance. The position of the primers used to confirm the correct integration are shown as arrows. The long-range PCR used to monitor the Zmym3tm(NOCOM)Mfgc allele is shown on the right. (C) Unimpaired in vitro differentiation of ZMYM3-/ ES cells into neuronal-like cells. In vitro differentiation of Zmym3-/ ES cells following treatment with retinoic acid compared to the C2 parental ES cell line. The time line of the retinoic acid treatment and the time points used for comparison are shown. ES cells were photographed at the times indicated using a Leica DMIL LED Microscope (Leica Microsystems) using a 5x objective, and a QIClick camera and QCapture Suite Plus version 3.1.3.10 (both QImaging).(A) Confocal micrographs of HA-tagged ZMYM family proteins. HEK293T cells imaged 24 hours post-transfection. Magnification: 60x. Antibodies used include Mouse Anti-HA and Alexa Fluor 568 Goat anti-Mouse. (B) Schematic illustration of the mouse (TIF)Click here for additional data file.S6 FigA schematic linear representation of ZMYM3 as a modular scaffold for an array of proteins involved in chromatin modification and recognition. The zinc finger 1 domain is involved in the interaction with General Transcription Factor IIi (GTFII-I) which can recognize DNA in a sequence specific manner (though binding to promoters containing Inr initiator and E-box motifs) whereas the KDM1A/CoREST/HDAC2 LCH complex associates with the central region of the protein through zinc fingers 8 and 9. The C-terminal portion of the protein can recruit RNase H2 to chromatin and DNA though the PXP proline rich domain. This provides a mechanism to coordinate histone tail modification by the LSD1/KDM1A demethylase, histone deacetylation by HDAC2 and transcriptional silencing by CoREST with RNA/DNA hybrid recognition and removal via the RNase activity.(TIF)Click here for additional data file.S7 FigThe source images used for each figure are shown and the regions selected to composed the corresponding figures are indicated with the same labelling as used in the main figures.(PDF)Click here for additional data file.


Type I interferon (IFN)-stimulated gene (ISG) expression requires interaction between a transcription factor complex, ISGF3, and target gene promoters to initiate transcription and protection against infection. To uncover chromatin regulatory features of this antiviral immune response, IFN-induced nucleosome and histone dynamics of human ISG loci were examined. ISGF3 recruitment after IFN stimulation was accompanied by nucleosome reorganization at promoters and gene bodies. IFN stimulation induced loss of core histones H2B, H3, and H4, as well as H2A.Z at ISG promoters. A strong correlation was found between H2A.Z occupancy and ISGF3 target sites, and IFN-stimulated H2A.Z removal requires STAT1, STAT2, and IRF9. Neither INO80 nor SWI/SNF participate in IFN-driven H2A.Z eviction, but GCN5 and BRD2 are required. Interference with H2A.Z expression enhanced ISGF3 recruitment to ISG promoters, ISG mRNA expression, and IFN-stimulated antiviral immunity. This indicates that H2A.Z nucleosomes at ISG promoters restrict optimal ISGF3 engagement and modulate the biological response to IFN.  •Type I IFN induces nucleosome reorganization at antiviral target genes•Histone H2A.Z occupies ISG promoters and is removed following IFN stimulation•IFN induces H2A.Z remodeling via STAT1, STAT2, IRF9, and cofactors GCN5 and BRD2•H2A.Z knockdown increases promoter access, ISG transcription, and antiviral response Immunology; Immune Response; Transcriptomics Initially discovered as a soluble antiviral factor secreted from infected cells , type I IFN actions are largely mediated by a canonical JAK-STAT signaling pathway that induces ISG products . IFN binDrosophila, Xenopus, Tetrahymena, and Trypanosoma  and targeted high-resolution nucleosome position analysis. An IFN-induced nucleosome loss observed at most of the surveyed promoters led to examination of the histone composition at these loci. Evidence is presented here indicating that ISG promoters, like many active eukaryotic promoters, are decorated with the variant histone H2A.Z. This variant nucleosome composition exhibits dynamic behavior in response to IFN stimulation, with loss and recovery coordinated with ISGF3 activity. The loss of H2A.Z requires ISGF3, the HAT, GCN5, and the “bromodomain and extraterminal” (BET) protein BRD2. Interference with H2A.Z enhances ISGF3 recruitment to ISG promoters and hyperactivates ISG expression, leading to greater IFN-induced antiviral protection in cells with H2A.Z depletion. These results reveal dynamic nucleosome remodeling associated with IFN-stimulated transcription and indicate a negative regulatory role for H2A.Z nucleosomes in innate antiviral immune signal transduction.OAS3 promoter was observed between 2 and 4 hr of IFN stimulation and was attenuated after 6 hr  remodeler subunits. Nucleosome occupancy at these loci was surveyed at steady state and after 2, 6, and 10 hr of IFN stimulation  using a direct selection micrococcal nuclease method to provide greater sequencing read depth per nucleosome  or +1 and NDR nucleosomes , those with only +1 nucleosomes (STAT2), or those with only −1 nucleosomes (OAS3), and genes with no apparent NDR . In all cases, IFN stimulation resulted in decreased nucleosome positioning strength, with clear disruption over the course of IFN treatment followed by a return to steady state. In most cases, this is evident from a loss of an ISRE-proximal nucleosome , and examination of target promoter regions indicates that peaks of H2A.Z deposition closely overlap with STAT2-binding ISRE sites  and INO80 . In loweTM3/1-8U , and theTM3/1-8U .To determine the machinery involved in IFN-induced, ISGF3-dependent H2A.Z loss, these transcriptional cofactors were examined using chemical inhibitor-, small interfering RNA (siRNA)-, or short hairpin RNA (shRNA)-based approaches  and S1. Both HAT and HDAC activities are required for positive regulation of ISG transcription. Two HATs, GCN5 (KAT2A) and CBP (CREBBP), interact with ISGF3 and participate in ISG transcription . HDACs aHistone H3 and H4 acetylation levels have been shown to be altered in response to IFN stimulation , and H2AThe uniform decoration of ISG promoters with H2A.Z, and its IFN-induced loss and recovery, suggested a potential role for H2A.Z in ISG regulation and biological activity. To investigate the impact of H2A.Z in IFN responses and ISG transcription, knockdown experiments were conducted in cells harboring shRNA against H2A.Z or a non-silencing control sequence. H2A.Z-shRNA reduced H2A.Z protein levels by 64%–67% e.g., A. ChIP aH2A.Z mRNA levels did not change due to IFN stimulation, indicating it is not itself an ISG, and shRNA expression resulted in a significant decrease in H2A.Z mRNA (70%–78%)  C. ISG mR70%–78%) D. For alThe IFN-stimulated transcriptional response is the primary cell-autonomous innate antiviral response that inhibits virus replication. To test the overall phenotypic impact of H2A.Z deficiency in the IFN response, a biological response assay was used to assess a role for H2A.Z in IFN-induced antiviral protection. H2A.Z-shRNA and control shRNA cells were stimulated with IFN for 9 hr to establish an antiviral state, then challenged with vesicular stomatitis virus (VSV) infection, overlayed with agarose, and viral plaques quantified. Virus replication was virtually identical in H2A.Z-shRNA or control shRNA cells in the absence of IFN stimulation, irrespective of H2A.Z depletion . In contTo better understand the contribution of chromatin and nucleosome dynamics to mammalian antiviral transcriptional regulation, the chromatin architecture and nucleosome organization of ISG loci were characterized to correlate IFN-mediated changes with the activation of ISGF3 components, STAT1, STAT2, and IRF9. Overall, stable steady-state nucleosome positions at ISG loci were rearranged by IFN stimulation, giving rise to a transient alteration in chromatin structure during the response. These alterations were particularly evident at ISGF3-ISRE promoter regions, supporting the notion that ISGF3-mediated recruitment of chromatin-modifying enzymes serves to remodel chromatin.Direct examination of the histone composition of ISG promoters not only confirmed the IFN-induced nucleosome loss but also identified an absence of histone H2A. In its place, the H2A variant H2A.Z was found to be enriched at or near the ISRE regions of most highly responsive ISGs before IFN stimulation. H2A.Z presence at ISG promoters was found to be tightly but inversely correlated with IFN-stimulated STAT2 occupancy, and IFN stimulation induced acute and transient loss of H2A.Z at ISG promoters coinciding with the cycle of ISGF3 activation, inactivation, and transcription attenuation. H2A.Z removal requires ISGF3 components STAT1, STAT2, or IRF9, indicating a role for ISGF3 in recruiting and coordinating machinery for H2A.Z nucleosome eviction. Identification of H2A.Z at ISG promoters is in agreement with the general paradigm of H2A.Z nucleosome association with active or inducible gene promoters . Here, HTo determine the factor(s) regulating H2A.Z loss, we examined a broad array of co-activators that were known to be associated with ISGF3 and ISG transcription, as well as those implicated previously in H2A.Z deposition or removal from other systems. In lower eukaryotes, SWR1 (SRCAP), was demonstrated to deposit H2A.Z, and knockdown of mammalian SRCAP did not alter IFN-induced H2A.Z removal. Although the INO80 remodeling complex is purported to be responsible for removing H2A.Z nucleosomes in lower eukaryotes , resultsIFIT2/ISG54 promoter. We postulate that this might indicate redundancy in HAT activities or reflect heterogeneity at individual ISG loci. GCN5 or GCN5-containing HAT complexes have been shown to acetylate histones H3, H4, and H2A.Z, and these acetylated lysines are in turn recognized by BET family protein bromodomains (Instead, the HAT GCN5 was identified as being required for ISG transcription and as an essential component of H2A.Z eviction. Inhibition of GCN5 using MB-3 generally inhibited the IFN-induced H2A.Z removal at the ISG promoters examined in both HeLa and 2fTGH cell lines, but a smaller effect of GCN5 inhibition was observed at the odomains . The BETodomains , and we odomains .H2A.Z is thought to influence nucleosome stability and positioning, and consequently alter the ability of activating or repressing factors to make stable or transient contact with DNA. This general property of H2A.Z nucleosomes can result in both positive and negative regulation, depending on gene-specific, tissue-specific, and/or context-specific transcriptional responses . The phyRegulating ISG transcription is critical for cellular antiviral responses and for subsequent immune responses, and chronic IFN signaling can lead to inflammatory and autoimmune diseases . DysreguAll methods can be found in the accompanying


Archaeal NSun6 homologs possess conserved catalytic residues, but have distinct differences in their RNA recognition motifs from eukaryotic NSun6s. Until now, the biochemical properties and functions of archaeal NSun6 homologs were unknown. In archaeon Pyrococcus horikoshii OT3, the gene encoding the NSun6 homolog is PH1991. We demonstrated that the PH1991 protein could catalyze m5C72 formation on some specific PhtRNAs in vitro and was thus named as PhNSun6. Remarkably, PhNSun6 has a much wider range of tRNA substrates than hNSun6, which was attributed to its tRNA substrate specificity. The mechanism was further elucidated using biochemical and crystallographic experiments. Structurally, the binding pocket for nucleotide 73 in PhNSun6 is specific to accommodate U73 or G73-containing PhtRNAs. Furthermore, PhNSun6 lacks the eukaryotic NSun6-specific Lys-rich loop, resulting in the non-recognition of D-stem region by PhNSun6. Functionally, the m5C72 modification could slightly promote the thermal stability of PhtRNAs, but did not affect the amino acid accepting activity of PhtRNAs.Human NOL1/NOP2/Sun RNA methyltransferase family member 6 (hNSun6) generates 5-methylcytosine (m As linker molecules between ribosomes and growing polypeptide chains, transfer RNAs (tRNAs) play a central role in protein synthesis. Recently, further functions of tRNAs have been identified in addition to translation . Multiplin vivo functions; however, archaeal tRNA modifications are indispensable (Ile(CAU) is essential for precise decoding  is a ubiquitous modification in DNA and different cellular RNAs, which is formed by site-specific methyltransferases (MTases). The NSun family, which contains an S-adenosyl-methionine (SAM)-dependent NOL1/NOP2/Sun (NSun) domain, is the main RNA:m5C MTase and comprises seven family members in eukaryotes. Among them, NSun2, NSun3 and NSun6 are primarily responsible for m5C modifications on tRNAs  (m(hNSun6) . Our pre(hNSun6) . The str(hNSun6) . HomologPyrococcus horikoshii OT3 (P. horikoshii), five genes encode putative RNA m5C MTases: PH1537, PH1374, PH1078, PH1991, and PH0851. Among them, PH1991 shares the highest sequence similarity (23.9% identity) with hNSun6, suggesting that PH1991 is the hNSun6 homolog. Interestingly, compared with hNSun6, the amino acid sequence of PH1991 is shorter and the predicted RNA recognition residues, especially the predicted PUA domain (residues 86–164) and RNA recognition motifs , are extremely divergent from eukaryotic NSun6s , Thermococcus litoralis NS-C . In contrast to hNSun6, which could only methylate four tRNAs with U73, PhNSun6 could catalyze m5C72 formation on eleven PhtRNAs harboring either U73 or G73. We further characterized the tRNA recognition elements for PhNSun6, which were quite different from those of hNSun6. To better understand the recognition mechanism of PhNSun6, we solved the crystal structures of PhNSun6 in the apo form and in complex with SAM, sinefungin , and S-adenosyl-homocysteine . Furthermore, we found that m5C72-modified PhtRNAs show slightly higher thermal stability than their unmodified counterparts, but this does not affect the amino acid accepting activity of PhtRNAs. Combined with previous work, we proposed an evolutionary model for eukaryotic and archaeal NSun6 and its tRNA recognition mechanism.In this study, we identified that PH1991 is a tRNA:m5C standard, dithiothreitol (DTT), guanosine monophosphate (GMP), sodium acetate (NaAc), Tris-base, SAH, benzonase, pyrophosphate, and phosphodiesterase I from Crotalus adamanteus venom were purchased from Sigma-Aldrich Co. LLC. . Bacterial alkaline phosphatase was obtained from Invitrogen . Tris–HCl, tryptone, yeast extract, isopropyl-D-thiogalactoside (IPTG), bovine serum albumin (BSA), MgCl2, NaCl, sodium phosphate monobasic, sodium phosphate dibasic, Ethylene diamine tetraacetic acid (EDTA), ATP, CTP, GTP, and UTP were purchased from Sangon Biotech . DNA fragment rapid purification and plasmid extraction kits were purchased from Yuanpinghao Biotech . KOD-plus mutagenesis kit and KOD-plus-neo Kit were from TOYOBO . Crystallization kits were from Hampton research . T4 DNA ligase, the ribonuclease inhibitor, and all restriction endonucleases were obtained from Thermo Scientific . [Methyl-3H] SAM (78.0 Ci/mmol) and [3H] L-serine were purchased from Perkin Elmer Inc. . [14C] l-threonine was obtained from Biotrend Chemicals . SAM was purchased from New England BioLabs . SFG was purchased from Santa Cruz Biotechnology . PCR primers were synthesized by BioSune . The plasmid pTrc99b was from the Institut de Biologie Moléculaire et Cellulaire du CNRS . The pET28a vector was from Merck–Millipore . Escherichia coli Rosetta (DE3) cells were purchased from TIANGEN . Nickel-nitrilotriacetic (Ni-NTA) Superflow resin was purchased from Qiagen, Inc. . The Superdex™ 200 column  was purchased from GE Healthcare . T7 RNA polymerase was purified from an overproduction strain in our laboratory (The mboratory .PhtRNAs genes were inserted into pTrc99b to construct pTrc99b-T7-PhtRNAs. Mutants of the PhtRNAThr(CGU) were obtained using the KOD-plus mutagenesis kit. All tRNAs were produced using in vitro T7 RNA polymerase transcription, as described previously  was chemically synthesized by BioSune and inserted into the pET28a vector using NdeI and NotI. Escherichia coli Rosetta (DE3) was transformed with the pET28a-PhNSun6 plasmid. Protein overproduction was induced by adding 0.2 mM IPTG into cells. After culturing for 12 h at 18°C, the cells were collected by centrifugation. PhNSun6 was purified by affinity chromatography on Ni-NTA Superflow resin according to the manufacturer's protocol. The wet cells (∼3 g) were suspended in 15 ml of 10 mM imidazole in buffer A  and sonicated on ice. Then, the sonicated crude extracts were heated at 65°C for 10 min. The crude extracts were centrifuged at 40 000 × g for 40 min to remove the debris and insoluble fractions. The supernatant was mixed gently with 0.5 ml Ni-NTA resin for 30 min at 4°C and then washed with 50 ml of 20 mM imidazole in buffer A to remove nonspecific binding proteins. Binding proteins were eluted in 8 ml of 250 mM imidazole in buffer A. The eluted protein was concentrated and purified by gel filtration on a Superdex™ 200 column.The gene encoding PH1991  was mixed with an equal volume of the reservoir solution, consisting of 0.2 M magnesium acetate tetrahydrate, 0.1 M sodium cacodylate trihydrate pH 6.5 and 20% (w/v) polyethylene glycol 8000. PhNSun6 and 1 mM SAM, or SFG, or SAH, were co-crystallized under the same conditions. The crystals were frozen in liquid nitrogen after transferring for a few seconds in the mother liquor, which contained 15% (v/v) glycerol as a cryoprotectant.Crystallization was performed at 16°C by the hanging drop vapor diffusion method. For crystallization, PhNSun6 in the apo form was initially solved by molecular replacement using PHASER . Figures were drawn using PyMOL (http://www.pymol.org/). A structure-based multiple amino acid sequence alignment of NSun6s from model organisms was generated using ESPript  BL-19U1. The diffraction data were processed using the HKL3000 program package . Furtherg PHASER  with hNSg PHASER . The strIX suite . The qua ESPript .PhNSun6 and its mutants, 5 μM of tRNAs were used as substrates. The reactions were performed at 65°C under the same conditions in a 25-μl reaction mixture containing 200 μM 3H-SAM, 50 mM Tris–HCl (pH 7.5), 200 mM NaCl, 10 mM MgCl2, 100 μg/ml BSA, and 5 mM DTT. Reactions were initiated by the addition of 100 nM PhNSun6. Aliquots (5 μl) of the reaction mixtures were removed at time intervals between 2 and 8 min, quenched on Whatman glass-fiber filter discs, and soaked in 5% trichloroacetic acid (TCA). After washing, the amount of radioactive [3H]-methyl-tRNA on each disc was measured in a Beckman Las6500 scintillation counting apparatus. The steady-state kinetics were measured under the same conditions with 50–300 nM PhNSun6 and 0.1–40 μM tRNA; the reaction time was 2 or 4 min. For mutants that exhibited extremely low activity, higher concentrations of enzyme and tRNA substrates were used. The data were fitted to Lineweaver-Burk plots, and the Km, kcat and kcat/Km values for each tRNA were calculated from the curve fitting.To measure the methyl transfer activity of 5C72_PhtRNAs, the reactions were performed at 65°C in a mixture comprising 2 μM PhNSun6, 200 μM SAM, 50 mM Tris–HCl (pH 7.5), 200 mM NaCl, 10 mM MgCl2, 100 μg/ml BSA, and PhtRNAs. The wild-type (WT) PhtRNAs were prepared in the same mixture by adding the PhNSun6 restored buffer. The reaction was performed for 1 h and stopped using phenol/chloroform extraction. The PhtRNAs were precipitated using a 3-fold volume of ethanol.To obtain mPhtRNASer was determined in a 25-μl mixture containing 60 mM Tris–HCl (pH 7.5), 10 mM MgCl2, 5 mM DTT, 2.5 mM ATP, 100 μg/ml BSA, 40 μM [3H] serine, and 200 nM enzyme, with 5 μM of PhtRNASer at 60°C. Aliquots (5 μl) of the reaction mixtures were removed at time intervals between 1 and 4 min, quenched on Whatman glass-fiber filter discs, and soaked in 5% TCA. The time course curve for aminoacylation by ThrRS for PhtRNAThr was determined at 37°C, as described previously . The nucleosides were separated using UPLC on a C18 column  and then detected using a triple-quadruple mass spectrometer (Agilent 6400 QQQ) in the positive ion multiple reaction-monitoring (MRM) mode. Mass transition from m/z 258 to 126 (m5C) was monitored and recorded.One microgram of tRNA was hydrolyzed using benzonase, phosphodiesterase I, and bacterial alkaline phosphatase overnight at 37°C in a 20-μl solution including 20 mM Tris–HCl (pH 8.0), 2 mM MgClTm buffer . The initial absorbance of the tRNA at 260 nm was controlled between 0.2–0.3. A melting temperature curve was measured at 260 nm using a heating rate of 1°C/min from 25 to 95°C via an Agilent Cary 100 spectrophotometer. The Tm value was determined from the first derivative of the melting curve.Specific tRNAs were dissolved in P. horikoshii. To validate whether PH1991 has the tRNA:m5C72 MTase catalytic activity, we synthesized the gene that encodes PH1991 and prepared possible tRNA substrates: PhtRNAThr(CGU), PhtRNAThr(GGU), PhtRNAThr(UGU) and PhtRNACys(GCA), corresponding to the tRNA substrates of hNSun6, using T7 transcription.PH1991 is the homolog of eukaryotic NSun6 in PhtRNAThr(CGU), we tested the methyl transfer activity at 37, 55 and 65°C. The highest methyl transfer activity of PH1991 was at 65°C (PhtRNAThr isoacceptors and PhtRNACys(GCA) at 65°C, similar to hNSun6 at 37°C  methylated by PH1991 were analyzed using UPLC-MS and the modification was confirmed to be m5C , PhtRNAThr(UGU), and PhtRNACys(GCA) (PhtRNAThr(CGU)-C72A, -C72G, and -C72U, were constructed. None of these mutants could be methylated by PhNSun6 . Thus, we identified that PH1991 is the P. horikoshii tRNA:m5C72 MTase NSun6 and named it PhNSun6.Subsequently, we purified PH1991 and subjected it to SDS-PAGE analysis Figure . To assa at 65°C . We founC Figure . The PhtC Figure , as it wCys(GCA) . To conf6 Figure . We measbstrates . The Km PhNSun6. We assayed the methylation of a PhtRNAThr(CGU) mutant with a deleted CCA terminus and several mutations in U73 and C2:G71  mutant lacking the CCA terminus -U73A, -U73C and -U73G mutants -U73G, but not PhtRNAThr(CGU)-U73A or -U73C -U73G was 0.59 μM, which was slightly higher than that for PhtRNAThr(CGU) (0.43 μM). Whereas the kcat value of PhNSun6 for PhtRNAThr(CGU)-U73G was 2.11 min−1, which was almost a half that determined for PhtRNAThr(CGU) (4.30 min−1) (PhNSun6-mediated methylation of C72 of PhtRNAThr(CGU)-U73G is lower than that for PhtRNAThr(CGU). However, unlike hNSun6, which only recognizes U73, PhNSun6 can recognize both U73 and G73.To determine whether U73 was necessary for methylation by s Figure . RemarkaC Figure . Steady-0 min−1) . These rPhtRNAs, and in few PhtRNAs is G2:U71; the third base pair exists as C3:G70, G3:C70, A3:U70 and G3:U70. In the four above-mentioned PhtRNAs, the second and third base pairs are C2:G71 and C3:G70, respectively. To determine whether PhNSun6 recognized the second base pair, two mutants, PhtRNAThr(CGU)-C2G:G71C and -C2G:G71U, were constructed and their methylation by PhNSun6 was assessed . Unexpectedly, PhNSun6 could methylate all these mutants  mutants by replacing C11:G24 or C12:G23 with A:U, U:A, G:C, G:U and U:G, respectively   .PhNSun6.These results suggested that C11:G24 and C12:G23 base pairs in the D-stem are not involved in RNA recognition by PhNSun6 requires fewer identity elements for tRNA substrates than hNSun6. To examine whether PhNSun6 has other tRNA substrates besides the above four PhtRNAs (three PhtRNAThr isoacceptors and PhtRNACys(GCA)), we determined the methylation activity of PhNSun6 for all the PhtRNAs that harbor the recognition elements of PhNSun6. These PhtRNAs are PhtRNASer(UGA), -(CGA), -(GGA) and -(GCU), PhtRNAAsn(GUU), PhtRNAAsp(GUC) and PhtRNAArg(GCG). In addition, PhtRNAPhe(GAA) with A73 and G2:C71, which does not match the recognition requirements for PhNSun6, was chosen as a negative control   Figure .PhNSun6 could efficiently catalyze m5C72 formation on eleven PhtRNAs, which suggested that PhNSun6 has a wider range of tRNA substrates than hNSun6.Thus, the results showed that PhNSun6, we obtained the crystal structures of PhNSun6 with and without its cofactors, including apo-PhNSun6, and PhNSun6 complexed with SAM, SFG and SAH, which diffracted to 2.6, 2.5, 2.5 and 2.2 Å, respectively  and a Rossmann-fold catalytic core (residues 185–389), and the PUA domain (residues 86–164). The PUA domain is inserted into the MTase domain via two linkers: Residues 79–86 and residues 164–173  in the surface of MTase catalytic core   . In thisp Figure , which iPhNSun6 recognition. According to the PhNSun6/tRNA model, all the three nucleotides of the CCA terminus interact extensively with residues from the PUA domain and linkers  of PhNSun6 to Ala. All the three mutants showed reduced methyltransferase activities compared with WT protein. The Km values of PhNSun6-N98A and -E174A for PhtRNAThr(CGU) were ∼2-fold higher than that of PhNSun6, while their kcat values remain similar  was 5.3-fold higher than that of PhNSun6 while its kcat value decreased , -(GGA), -(UGA) and -(GCU), PhtRNAThr(CGU), -(GGU) and -(UGU)) of the eleven PhtRNAs. The aminoacylation activity of aaRS for the m5C72-modified PhtRNAs showed no obvious difference compared that for their unmodified counterparts , the Tm values for the other ten PhtRNAs with the m5C72 modifications all increased compared with those for their unmodified counterparts. However, the delta Tm values are between ∼1 and 2°C in most cases. Only PhtRNAAsp(GUC) showed a 3°C difference between methylated and unmethylated tRNAs. The results suggested that m5C72 contributes thermal stability towards tRNA, although the effect from this single modification is small.To determine whether there is a correlation between the mBased on this work and our previous work on eukaryotic NSun6 ,34, a rePhNSun6. Considering the stability of hctRNAs, we tested the methyl transferase activity of PhNSun6 to hctRNAThr(AGU), hctRNAThr(UGU), and hctRNACys(GCA) at 65 or 55°C. The results showed that the tRNA substrates of hNSun6 could not be methylated by PhNSun6 at 65°C , -(CGA), -(GGA) and -(GCU), PhtRNAAsn(GUU), PhtRNAAsp(GUC) and PhtRNAArg(GCG) should not be methylated by hNSun6. Our previous study showed that the tRNACys(GCA)-G2C:C71G mutant is not a substrate of hNSun6 (PhtRNAs: PhtRNAThr(CGU), -(GGU) and -(UGU), and PhtRNACys(GCA) with the C2:G71 base pair could not be methylated by hNSun6 for this reason. These results rationalized the identified tRNA recognition elements for PhNSun6 and hNSun6  the 3′ CCA terminus, (ii) the target site C72, (iii) U73 or G73, and (iv) the discriminatory base pair C2:G71 at the acceptor stem. The CCA terminus and the target site C72 are common recognition elements for PhNSun6 and hNSun6. However, they exist in almost all tRNAs, which are not enough to distinguish substrate tRNAs of NSun6. U73/G73 and C2:G71 function as discriminatory elements to identify PhNSun6 substrate tRNAs from nonsubstrate tRNAs.In this study, we identified that PH1991 is the PhNSun6/tRNA, the binding pocket of site 73 is capable of accommodating both U and G, which was in line with our methylation experiment in which eleven PhtRNAs with U73 or G73 could be substrates of PhNSun6. In the model, the base moiety of G71 was not in contact with PhNSun6. However, based on our results, C2:G71 functions as a discriminatory base pair that can select substrate tRNAs from other PhtRNAs. The exact recognition mechanism of PhNSun6 for C2:G71 requires further mechanistic and structural studies.In the superimposition model of PhNSun6/tRNA superimposition model, PhNSun6 does not interact with the D-stem region of the tRNA, because of the lack of the eukaryotic NSun6-specific Lys-rich loop, which is consistent with biochemical data on the effect of the D-stem on activity of PhNSun6. Thus, our biochemical and crystallographic data suggest that PhNSun6 ignores the sequential specificity of the D-stem of PhtRNA substrates. Considering that archaeal NSun6 homologs share high identity with each other, we speculated that the tRNA substrate specificity of PhNSun6 is conserved in other archaeal NSun6 homologs.Based on the 5C catalytic core to bind RNA substrates, resulting in the formation of NSun6’s last universal common ancestor (LUCA). Specifically, for the recognition by NSun6’s LUCA of tRNA substrates, the PUA domain recognized the common CCA terminus; the RRM recognized site 73; and the m5C catalytic core recognized C72 and base pairs near site 72. Later in evolution, NSun6 divided into two branches: Archaeal NSun6 and eukaryotic NSun6. The divergence of the RRM and PUA domain during evolution, which increased the precision and accuracy of RNA substrate discrimination, archaeal and eukaryotic NSun6 exhibit different discrimination mechanisms for its cognate tRNA substrates. For RNA substrate discrimination, eukaryotic NSun6 recognizes the acceptor stem and D-stem, while archaeal NSun6 only interacts with the acceptor stem. The evolutionary difference between archaeal and eukaryotic NSun6 has led to diverse cognate tRNA recognition mechanisms.Based on the results of this study, together with previous findings on hNSun6, we proposed an evolutionary model for NSun6 Figure . Origina5C modifications in RNA has been observed across the three domains of life. However, the tRNA:m5C modification is present only in archaea and eukaryotes (5C modification could increase hydrophobicity and reinforce stacking interactions, probably leading to tRNA stabilization (Saccharomyces cerevisiae, the m5C40 modification of tRNAPhe stabilizes the tRNA anticodon stem (5C48 and m5C49, together with m7G46, cause the rapid decay of certain tRNAs (The presence of mkaryotes ,32,53. Tlization . In Saccdon stem ; lack ofin tRNAs .5C72 modification does not affect the amino acid accepting activity of tRNAs, but slightly increases the thermal stability of PhtRNAs. Acceptor stem of tRNA is rarely modified, and m5C72 is one of the few modifications in this region. It remains possible that m5C72 has other biological functions in vivo, which awaits further studies on both archaeal and eukaryotic NSun6s.In archaea, NSun6s are present only in hyperthermophilic species . In termPhNSun6 have been deposited with accession code 5ZVD; for the PhNSun6-SAM, PhNSun6-SFG, and PhNSun6-SAH complex under accession code 5ZVG, 5ZVH and 5ZVE, respectively.Protein Data Bank: atomic coordinates and structure factors for apo Supplementary DataClick here for additional data file.


The intracellular and extracellular pH of cancer cells affects their growth; however, the role of ATP6V0C in highly invasive esophageal cancer cells (ECCs) remains unclear. In this study, we examined the role of ATP6V0C in glucose metabolism in ECCs. The ATP6V0C depletion attenuated ECC proliferation, invasion, and suppressed glucose metabolism, as indicated by reduced glucose uptake and decreased lactate and adenosine triphosphate (ATP) production in cells. Consistent with this, expression of glycolytic enzyme and the extracellular acidification rate (ECAR) were also decreased by ATP6V0C knockdown. Mechanistically, ATP6V0C interacted with pyruvate kinase isoform M2 (PKM2), a key regulator of glycolysis in ECCs. The ATP6V0C depletion reduced PKM2 phosphorylation at tyrosine residue 105 (Tyr105), leading to inhibition of nuclear translocation of PKM2. In addition, ATP6V0C was recruited at hypoxia response element (HRE) sites in the lactate dehydrogenase A (LDHA) gene for glycolysis. Thus, our data suggest that ATP6V0C enhances aerobic glycolysis and motility in ECCs.The vacuolar H Esophageal cancer is one of the most lethal malignancies in the world . In the 18F] fluoro-D-glucose (FDG) have revealed excessive glucose accumulation within human esophageal tumors [Cancer cells constitutively activate glucose metabolism to promote cell survival and proliferation . Throughl tumors , indicat+-adenosine triphosphatase (ATPase) (V-ATPase), a large multi-subunit enzyme, becomes associated with mTOR to enhance mTORC1 translocation to the lysosomal surface [Glucose and amino acids are critical nutrients for cancer cell metabolism and growth ,8. Gluco surface . In addi surface , indicat2 and lactic acid production, which is required for cancer cell growth [Vacuolar ATPase plays an important role in pH homeostasis by regulating intensive respiratory COl growth . Vacuolal growth . On the l growth . Of all l growth . Consistl growth . Moreovel growth . HoweverIn this study, we investigated the effect of ATP6V0C depletion on cell survival, migration, invasion, and aerobic glycolysis in ECCs. We also analyzed how ATP6V0C regulates aerobic glycolysis in ECCs.Both TE8 and TE1 cells  were cultured in RPMI-1640 Medium  containing 10% fetal bovine serum . The SH-SY5Y ; HeLa ; SK-MEL-1 ; and U2OS cells  were cultured in DMEM  containing 10% fetal bovine serum . Immortalized human esophageal normal fibroblasts (ENFs) were obtained from Seok-Hyung Kim , a specialized gastrointestinal pathologist. He grossly examined esophageal tumor tissue and obtained samples of distal normal tissue . The ENFThe RNA purification and quantitative reverse transcriptase polymerase chain reaction (qRT-PCR) were performed, as described previously . Table SAliquots of the TE8, TE1, SH-SY5Y, HeLa, SK-MEL-1, and U2OS cell suspensions were mixed with trypan blue dye and were left for 5 min at room temperature. Then, the TE8, TE1, SH-SY5Y, HeLa, SK-MEL-1, and U2OS cells were counted using a hemocytometer and the percentage of live cells was determined.Western blotting was performed, as described previously , using a4 cells/well and cultured for 18 h. After incubation, the filters were removed from the chambers, the TE8 cells that had invaded the Matrigel were fixed and were stained with hematoxylin and eosin (H&E), and the number of TE8 cells attached to the filters was counted under a light microscope. A migration assay was conducted in a similar manner as the invasion assay, except that the filters were not coated with Matrigel. All assays were repeated a minimum of two times.An in vitro Matrigel  invasion assay was conducted using 8.0 µm (pore-size of inserts) Costar transwell chambers . The transwell filters were coated with Matrigel (1 mg/mL). The TE8 cells were transfected with non-silencing (NS) or ATP6V0C siRNA for 72 h, and TE8 cells were then seeded again onto Matrigel at the same density of 5 × 10Precleared TE8 cell lysates were incubated with protein G Sepharose beads and primary antibodies or immunoglobulin G  overnight at 4 °C. The tubes were then centrifuged, and the supernatant was removed. The lysates were rinsed three times in PBS containing protease inhibitor. The proteins were eluted from the beads by adding 5× sample loading buffer. After centrifugation, Western blots were performed on the samples to analyze the precipitation of proteins.Purified GST-PKM2 fusion protein and GST control (20 μg) were separately mixed with 20 μL of pre-equilibrated GST-agarose  in sodium chloride tris-ethylenediaminetetraacetic acid (EDTA) (STE) buffer. The mixtures were incubated overnight at 4 °C with agitation and then washed thrice with STE buffer before elution with 50 mM reduced glutathione . Protein samples from total cell extracts, immunoprecipitation (IP), GST pull-down, and chemical cross-linking were separated by 10% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), followed by Western blotting with antibodies, as indicated.The TE8 cells were blocked with bovine serum albumin (BSA) for 1 h. Then, the TE8 cells were immunostained using antibodies against ATP6V0C (cat. NBP1-31492), PKM2 (cat. 4053), Calnexin (cat. sc46669), Lamin B (cat. sc6217), Cox-1 (cat. 459600), and p-paxillin . Next, cells were treated with an anti-rabbit fluorescein isothiocyanate (FITC)-conjugated secondary antibody (Invitrogen), an anti-mouse rhodamine-conjugated secondary antibody (Santa Cruz Biotechnology), and an anti-goat rhodamine-conjugated secondary antibody (Invitrogen) or Alexa 488 phalloidin (Invitrogen) containing 1 mg/mL Hoechst (Invitrogen); then, they were mounted with anti-fading mounting medium . The TE8 cells were then visualized, and images were collected using fluorescence microscopy .TM ADP/ATP Ratio Assay kit , respectively, according to the manufacturers’ instructions. The total number of cells was used for normalization.Lactate levels and ATP production in TE8 cell lysates were measured using a colorimetric L-Lactate Assay kit  and an EnzyLight5 cells/well for 24 h. Then, the TE8 cells were transfected with NS or ATP6V0C siRNA using G-fectin (GP-2000) under high- or low-glucose conditions. For the glucose uptake assay, all of the culture medium was removed from each well and replaced with 2 mL of the culture medium in the absence or presence of 10 μM fluorescent 2-deoxy-2- amino)-D-glucose (2-NBDG). Then, the TE8 cells were incubated for 1 h at 37 °C with 5% CO2 before flow cytometry analysis. After removal of the incubation medium, the TE8 cells were washed twice with ice-cold PBS. This PBS (500 μL) was subsequently used to resuspend the cells. Then, 1 μg/mL of propidium iodide (PI) was added, and the TE8 cells were maintained at 4 °C for flow cytometry analysis using the BD FACS Canto II system .The TE8 cells were plated in 6 well plates at a density of 2 × 106 cells/mL. One hundred microliters of resuspended cells were transferred to a 5 mL tube, and 5 μL of FITC Annexin V and 5 μL PI were added. The cells were gently vortexed and incubated for 15 min at room temperature (25 °C) in the dark. Four hundred microliters of 1× binding buffer was then added to each tube, and flow cytometric analysis performed within 1 h.The ENFs were washed twice with ice-cold PBS and then resuspended in 1× binding buffer to a concentration of 10p-(trifluromethoxy) phenylhydrazone (FCCP) (uncoupler of oxidative phosphorylation in mitochondria) was added to determine mitochondria respiration capacity. Finally, 100 nM rotenone  was added to inhibit mitochondrial electron transport chain (ETC).The extracellular acidification rate (ECAR) and the oxygen consumption rate (OCR) of cultured TE8 cells were measured using a Seahorse XF24 extracellular flux analyzer according to the manufacturer’s instructions. Briefly, 25 mM glucose was added to stimulate cellular extracellular acidification and oxygen consumption, and 1 mM oligomycin (inhibitor of ATP synthase) was added to measure the glycolytic ability or ATP-linked OCR; 300 nM carbonyl cyanide To prepare cytosolic and nuclear lysates, TE8 cells were lysed in buffer A (10 mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES), pH 7.9; 10 mM KCl; 0.1 mM EDTA; 1 mM dithiothreitol (DTT); 0.5% NP-40; and protease inhibitors) and incubated on ice for 15 min. The cytosolic fractions were harvested by centrifugation at 13,000 rpm for 2 min at 4 °C. Next, the nuclear pellets were washed with ice-cold PBS to avoid the mixing of cytosolic proteins, lysed in buffer B , and incubated on ice for 10 min. The nuclear fractions were harvested by centrifugation at 13,000 rpm for 10 min at 4 °C.The TE8 cells were seeded onto 10 cm dishes in RPMI 1640 Medium 1 day before treatment. The TE8 cells were incubated in RPMI 1640 Medium containing 25 mM glucose for 6 h, as previously described . A chromt-tests. Differences among individual group means were analyzed by ANOVA or two-tailed t-tests. All assay data were presented as mean ± standard error of the mean (SEM). A two-sided significance level of 0.05 was used for all statistical analyses.The viability, migration, and invasion assay results were analyzed using one-way analysis of variance (ANOVA), two-way ANOVA, or Student’s The ATP6V0C is essential for maintaining the pH gradient between the cytoplasmic and lysosomal compartments . The intCell proliferation was markedly reduced B and theTo determine whether the reduction of ATP6V0C levels is toxic to ENFs, we used siRNAs to deplete ATP6V0C and evaluate the effect on the cells. The proliferation of ENFs was unaffected by ATP6V0C depletion when compared to cells treated with NS .In exploring whether ATP6V0C regulates the expression of cell-cycle regulators and cell survival-related proteins in TE8 cells, we found that reducing levels of ATP6V0C decreased the expression of cyclin E, cyclin-dependent kinase 2 (cdk2), and b-cell lymphoma (bcl-2), indicating G1 phase arrest D. In addWe further investigated whether ATP6V0C depletion also affects the proliferation of other cancer cell lines. The ATP6V0C depletion led to a reduction in proliferation of neuroblastoma SH-SY5SY, cervix adenocarcinoma HeLa, melanoma SK-MEL-1, and osteosarcoma U2OS cells . InteresGiven that most cancer cells use glucose as the main carbon source for anabolic growth , we examThese results suggested that ECC viability is sensitive to glucose availability and ATP6V0C depletion results in a decrease in the survival of ECCs; this was not observed with ENFs.Maintenance of intracellular pH homeostasis is essential for cancer cell motility leading to metastasis, and an acidic environment is enhanced by proton-sensing proteins . TherefoInhibition of glycolysis using 2-deoxy-D-glucose (2DG) suppresses the metastatic phenotype in colorectal cancer . TherefoGlucose metabolism facilitates cancer cell proliferation by increasing the synthesis of ATP and lactate, which are the final products of glycolysis . TherefoGlut1), hexokinase II (HK2), and lactate dehydrogenase A (LDHA), in aerobic glycolysis [Glut1, HIF-1, HK2, phosphofructokinase-1 (PFK1), enolase 1 (ENO1), PKM2, LDHA, and pyruvate dehydrogenase lipoamide kinase isozyme 1 (PDK1), under both high- and low-glucose conditions (Glucose uptake enhances the expression of hypoxia inducible factor 1 (HIF-1) target genes, such as glucose transporter 1  and tyrosine residue 148 (Tyr148) [The PKM2 domain containing amino acids 1–116 includes catalytic active sites, such as tyrosine residue 105 (Tyr(Tyr148) , which a105) of PKM2 increases PKM2 activity, leading to enhanced aerobic glycolysis [Tyrosine phosphorylation , in TE8 cells (LDHA HRE site.We investigated using the ChIP assay whether ATP6V0C stimulates PKM2 recruitment to hypoxia response element (HRE) sites of E8 cells C, suggesTo determine the clinical relevance of ATP6V0C expression in esophageal cancer, we retrieved whole exome sequencing and mRNA expression data from 10,967 patients with cancers from The Cancer Genome Atlas (TCGA) database. Genetic changes in ATP6V0C, particularly its deletion or amplification, were frequently found in esophageal cancer, underscoring the significance of ATP6V0C in tumors D. The reTogether, these results suggest that ATP6V0C induces the nuclear translocation of PKM2, leading to enhanced transcriptional activation of glycolytic genes, and eventually resulting in stimulation of glycolysis in ECCs E.The vacuolar-ATPase is composed of a cytosolic V1 domain for ATP hydrolysis and a transmembrane V0 domain, which drives proton translocation across the membrane . The vacOur analysis of TCGA data further confirmed that genomic alteration of ATP6V0C is frequently observed in ESCC, suggesting its clinical significance in esophageal squamous cancer. Consistent with our findings, bafilomycin, concanamycin, and archazolid, which decrease ATP6V0C activity, inhibit the growth and proliferation of breast, prostate, and leukemia cancer cells respectively ,44,45, sOur study further demonstrated that ATP6V0C positively regulates glucose metabolism, which produces main energy sources for esophageal cancer cells. Lowering levels of ATP6V0C resulted in reduced glucose uptake and decreased lactate and ATP production under both high- and low-glucose conditions, leading to downregulation of glycolysis. These results suggested that ATP6V0C might prevent cell death by enhancing glycolysis under low-glucose conditions.ATP6V0C knockdown cells after treatment with mitochondrial inhibitors, suggesting that ATP6V0C is crucial for glycolysis in ECCs.We assessed further how ATP6V0C regulates aerobic glycolysis to maintain cell proliferation under low-glucose conditions. In this study, ATP6V0C expression was increased further under low-glucose conditions. Consistent with this, ATP6V0C depletion prevented low-glucose-induced expression of glycolytic enzymes. In addition, ATP6V0C depletion decreased the ECAR, an indicator of aerobic glycolysis, whereas there was no significant difference in OCR between the NS and The ATP6V0C depletion inhibits the expression of glycolytic enzymes and decreases glycolysis, the first step in glucose oxidation. Studies have revealed that glucose deprivation contributes to mutations in the KRAS pathway  and induDuring glycolysis, pyruvate kinase (PK), which catalyzes the transfer of a phosphate from phosphoenolpyruvate to pyruvate, contributes to active glucose metabolism in cancer cells . While PThe molecular mechanisms underlying the interaction of ATP6V0C with PKM2 in TE8 cells are unclear. Our findings revealed that ATP6V0C associates preferentially with the PKM2 mutant R399E compared to WT PKM2. The PKM2 mutant R399E exists as a dimer and appears to be catalytically inactive for the conversion of phosphoenolpyruvic acid (PEP) to pyruvate . The inaThe assembly of the vacuolar H+-ATPase complex occurs when high levels of glucose are available, whereas glucose depletion induces V-ATPase disassembly leading to the inhibition of V-ATPase activity ,55. Inte105. In addition, ATP6V0C depletion resulted in inhibition of PKM2 nuclear translocation. Relevant to this, previous studies have reported that erythroblastic oncogene B, a receptor tyrosine kinase 2, induces PKM2 phosphorylation at Tyr105 by increasing nuclear localization of Yes-associated protein, and enhances cancer cell-like properties [105. In addition, the PKM2 Y105F mutation leads to a reduction in lactate production and an increase in oxidative phosphorylation [105. Our results suggest that ATP6V0C indirectly affects phosphorylation of PKM2 during glycolysis. Considering both that an increase in PKM2 phosphorylation at Tyr105 is positively associated with nuclear localization of PKM2 [105, although the kinase responsible has not yet been identified in esophageal cancer cells.The ATP6V0C and PKM2 phosphorylation increased under low-glucose conditions. The ATP6V0C depletion led to a decrease in PKM2 phosphorylation at Tyroperties . The fibrylation , pointin of PKM2  and our LDHA, which catalyzes the intra-conversion of pyruvate and lactate to enhance glycolysis.The ChIP analysis revealed that ATP6V0C depletion results in reduced recruitment of PKM2 to HRE sites, the gene promoter for transactivation of 105 and leads to nuclear translocation of PKM2 to LDHA HRE sites, enhancing transcriptional activation of glycolytic genes and, thus, inducing metabolic reprogramming and movement of ECCs. Therefore, targeting ATP6V0C could be an effective therapeutic approach for controlling deregulated glucose metabolism in ECCs.In conclusion, glucose-sensitive ATP6V0C interacts with the catalytic domain of PKM2 containing Tyr


The prepared Mo2C QDs exhibited a strong near-infrared (NIR) absorbance with a high molar extinction coefficient of 4.424 Lg−1cm−1 at 808 nm, a high photothermal conversion efficiency of 42.9%, and showed excellent performance on photoacoustic imaging. The Mo2C QDs had high stability and highly biocompatibility, with low cytotoxicity. Under NIR irradiation, a remarkable in vitro and in vivo therapeutic effect was obtained. Such a stable and biocompatible all-in-one theranostic nanoagent generated by facile synthesis that combines promising imaging guidance and effective tumor ablation properties may hold great potential for theranostic nanomedicine.Theranostic platforms integrating imaging diagnostic and therapeutic interventions into a single nanoplatform have attracted considerable attention for cancer-individualized therapies. However, their uncertain stability, complex pharmacokinetics, and intrinsic toxicology of multiple components hinder their practical application in clinical research. In this paper, stable and high-concentration molybdenum carbide quantum dots (Mo Nanotechnologies have played significant role in the development of both diagnostic and therapeutic agents and have contributed to the emergence of therapeutic examples expected to contribute to precise medicine ,4,5,6,7.Thermal ablation cancer therapy is a simple, safe, and minimally invasive treatment technique based on partial extreme temperature, which directly treats irreversible injury and coagulation necrosis of tumor cells ,22. Vari2C) belongs to the new family of MXenes and has attracted considerable interest due to its intriguing properties [2C—where the carbon atom layer is sandwiched between two molybdenum atoms layers by covalent forces—exhibits semimetal-like properties. On the one hand, it has high melting point, hardness, wear resistance, and oxidation resistance; on the other hand, it shows excellent electronics and optoelectronics activity for energy storage and catalysis [2C nanomaterials have never been explored.The two-dimensional (2D) ultrathin molybdenum carbide  were prepared through a facile liquid exfoliation method  effect. These Mo2C QDs were employed as nanotheranostic agents for photoacoustic (PA)/photothermal (PT) imaging-guided PTT for cancer.In this work, for the first time, water-soluble monolayered and high-concentration Mon method . The obt2C QDs were synthesized as previously reported, with some modification [2C powder was dispersed in 200 mL ultrapure water and sonicated (400 W) for 20 h at room temperature. After centrifugation at 5000 rpm for 15 min to remove the flakes of Mo2C, the resulting supernatant was filtered through a 0.22 µm microporous membrane , and the Mo2C QDs solution was obtained.The Mofication . Briefly2C QDs was measured by 3--2,5-diphenyl-2-H-tetrazolium bromide (MTT) assays on B16-F10 cells. B16-F10 cells were cultured for 12 h in a 96-well plate . 100 μg/mL Mo2C QDs ) or pure Opti-MEM were then added to the wells and co-cultured for 4 h. After incubation for another 8 h, the cells were exposed to an NIR laser  for 0–10 min and then incubated for another 24 h.In vitro photothermal effect of Mo5) transfected with Mo2C QDs were cultured in a confocal dish, irradiated with an 808 nm laser , and incubated with calcein-AM and PI solution for 30 min.Confocal imaging was performed with an Olympus FV1200 laser scanning confocal microscope . The B16-F10 cells . The Institutional Animal Care and Use Committee of the Beijing Institute of Basic Medical Science  approved all the animal experiments. Tumor-bearing nude mice were intravenously injected with Mo2C QDs solution, and PA signal was recorded on a multispectral optical tomography system . A series of Mo3, BALB/c tumor-bearing nude mice were divided into four groups (n = 5): The first group was intravenously injected with phosphate buffer saline  as control group; the second group was intravenously injected with Mo2C QDs in PBS; the third group was only irradiated by NIR laser (808 nm); and the fourth group was intravenously injected with Mo2C QDs and then exposed to 808 nm laser. Four hours after intravenous injection, the nude mice were anesthetized and irradiated with 808 nm laser . The tumor volume was calculated according to the formula: length × width2/2. Moreover, the tumor issue were sliced and stained for histological analysis.When the tumor volume reached ~100 mm2C powder was used as precursor to synthesize the monolayered Mo2C QDs. As shown in 2C powder clearly displayed a multilayer flat structure and the transmission electron microscopy  image of the Mo2C QDs indicated the monodisperse crystalline with a uniform diameter of ≈6 nm  and superior to graphene oxide nanosheets (3.6 Lg−1 cm−1) and gold nanorods (3.9 Lg−1 cm−1) at 808 nm [The UV-vis−NIR absorption spectra a obtainenosheets  and Ti3Cnosheets . It has ansition ,34,35. At 808 nm ,37. 2C QDs in the 700–850 nm inspired us to further investigate its potential application as promising photothermal and PA imaging agent. As shown in 2C QDs solution increased by 36 °C at low concentration (100 μg/mL), indicating that the Mo2C QDs could efficiently convert NIR light into thermal energy. The photothermal conversion efficiency (η) of Mo2C QDs was calculated to be 42.9% (3C2 nanosheets (30.6%) [2C QDs did not display obvious deterioration during the five heating–cooling cycles, highlighting the good photothermal stability and reproducibility of Mo2C QDs as a photothermal agent  . The phoal agent f. 2C QDs at various concentrations of Mo2C QDs, and the results  irradiation produced slight cytotoxicity to B16-10F cells in 10 min. By contrast, the Mo2C QDs-incubated B16-10F cells displayed high photocytotoxicity, and over 90% cells were killed after 10 min irradiation due to the photothermal effect of Mo2C QDs. The results of calcein AM  and propidium iodide  co-staining assay was consistent with of the MTT analysis . The excellent PTT efficiency of the Mo2C QDs is probably due to the high photothermal conversion efficiency and good transfection efficiency. In vitro cytotoxicity of the MoMo2C QDs a. Figureanalysis c. Neithe2C QDs as theranostic nanoagents. 2C QDs. As expected, negligible PA signal was detected at the tumor location before Mo2C QDs injection, while a strong PA signal was observed after injection, indicating the efficient tumor accumulation of Mo2C QDs through the enhanced permeability and retention (EPR) effect [2, 10 min), the tumor site temperatures of Mo2C QDs-treated mice increased from 27.9 to 63 °C, while the PBS-treated mice exhibited only a slight temperature increase.We further carried out in vivo experiments to test the feasibility of Mo) effect ,42. The 2C QDs was measured to evaluate their blood circulation time and metabolism. As shown in 2C QDs in bloodstream conformed well to the two-compartment model with a blood circulation half-time of 1.36 h. The quantification analysis shown in 2C QDs (12.26% ID g−1) through the EPR effect. The significant accumulation in major organs, including liver, spleen, and kidney, were also observed. The high level of Mo2C QDs in liver and spleen was possibly owing to mononuclear phagocytic system absorption, and the accumulation in kidney could be correlated with renal excretion.The pharmacokinetics profile and biodistribution of the Mo2C QDs were performed on B16-10F tumor-bearing nude mice, which were divided into four groups (n = 5): PBS injection; Mo2C QDs injection; PBS injection and 808 nm laser irradiation; and Mo2C QDs injection and 808 nm laser irradiation. The tumor volume and body weights of all these four group were measured as the function of time for 15 days  a and terst ki-67 ,44. Simist ki-67 ,45. The reatment d. In the2C QDs nanoagent using a facile sonication-assisted liquid-phase exfoliation method for PA/photothermal imaging-guided PTT without extra-complicated modification. The Mo2C QDs displayed an advanced extinction coefficient, high photothermal conversion efficiency, and excellent photothermal stability as a result of the semimetal-like energy band structure and the related strong NIR LSPR properties. Low cytotoxicity, good biocompatibility and physiological stability were also revealed. The Mo2C QDs achieved sensitive dual-modal PA/PT tumor imaging diagnosis and highly efficient tumor ablation in vitro and in vivo with negligible adverse effect. The work has explored the biomedical application of Mo2C QDs for the first time and will contribute to the design of single theranostic nanoparticles with intrinsic imaging and therapeutic capabilities.For the first time, we have synthesized an all-in-one semimetal, ultrasmall Mo


S100A7 gene, also called psoriasin, was first described as an upregulated protein in psoriatic skin. For the past years, the importance of this protein as a key effector of innate immunity has been clearly established, not only due to its importance protecting against bacteria skin insult in humans, but also because of its important role in amplifying inflammatory processes. Given the importance of S100A7 in host defense, S100A7 genes have been mostly studied in humans. Here we provide a detailed analysis of the evolution of the gene family encoding for the S100A7 protein in mammals.The S100A7. Among the most representative mammalian groups, we report that multiple events of duplication, gene loss and high mutation rates are shaping the evolution of this gene family. An unexpected result comes from Myotis species (order Chiroptera), where we found an outstanding S100A7 gene radiation, resulting in more than 10 copies in M. lucifugus and 5 copies in M. brandtii. These findings suggest a unique adaptive road in these species and are suggestive of special role of this protein in their immune system.Examination of several mammalian genomes revealed an unexpected variation in the copy number of S100A7 within mammals and therefore will pave the way to further clarify their unique function in the immune system.We found different evolutionary histories among different mammalian groups. Overall, our results suggest that this gene family is evolving under the birth-and-death model of evolution. To our knowledge, this work represents the first detailed analysis of phylogenetic relationships of The online version of this article (10.1186/s12862-019-1433-0) contains supplementary material, which is available to authorized users. S100A7 expression occur in response to several cytokines, including IL-17, IL-22 and TNF (Tumor Necrosis Factor)  , 67. TheAdditional file 1:S100A7 genes used in this study. All sequences are available from NCBI and Ensembl databases. (PDF 109 kb)List of the sequences of the Additional file 2:B. acutorostrata_A7(1) coding sequence. (PDF 275 kb)Alignment of S100A7 coding sequences from several eutherian mammals. The abbreviations correspond to the ones shown in Additional file Additional file 3:P. troglodytes and two S100A7 proteins from H. sapiens and P. paniscus. Dots = identity with M. musculus_S100A7_XM_ 006501635.3 protein. (PDF 305 kb)Alignment between S100A15 and S100A7 proteins. Alignment between two S100A15 proteins from mouse, one S100A15 from Additional file 4:Mus musculus S10015 protein. (PDF 172 kb)Alignment of S100A15 proteins from several eutherian mammals. Dots = identity with Additional file 5:S100A7 and S100A15 from mouse and human, respectively. (PDF 193 kb)Search for the gene loss remnants of Additional file 6:M. lyra - Megaderma lyra; R. ferrumequinum - Rhinolopus ferrumequinum; R. sinicus - Rhinolophus sinicus; P. patnellii - Pteronotus parnellii; H. arimiger - Hipposideros armiger; D. rotundus - Desmodus rotundus. Dots = identity with M. lucifugus_A7(1) sequence. (PDF 149 kb)Alignment of partial sequences S100A7 from bats. The abbreviations correspond to the following species: Additional file 7:Selection (dN/dS) analyses. (PDF 331 kb)


Saccharomyces cerevisiae gene SEC53. Here we describe conserved genotype-phenotype relationships across yeast and human patients between five PMM2 loss-of-function missense mutations and their orthologous SEC53 mutations. These alleles range in severity from folding defective (hypomorph) to dimerization defective (severe hypomorph) to catalytic dead (null). We included the first and second most common missense mutations – R141H, F119L respectively– and the most common compound heterozygote genotype – PMM2R141H/F119L – observed in PMM2-CDG patients. Each mutation described is expressed in haploid as well as homozygous and heterozygous diploid yeast cells at varying protein expression levels as either SEC53 protein variants or PMM2 protein variants. We developed a 384-well-plate, growth-based assay for use in a screen of the 2,560-compound Microsource Spectrum library of approved drugs, experimental drugs, tool compounds and natural products. We identified three compounds that suppress growth defects of SEC53 variants, F126L and V238M, based on the biochemical defect of the allele, protein abundance or ploidy. The rare PMM2 E139K protein variant is fully functional in yeast cells, suggesting that its pathogenicity in humans is due to the underlying DNA mutation that results in skipping of exon 5 and a nonfunctional truncated protein. Together, these results demonstrate that yeast models can be used to characterize known and novel PMM2 patient alleles in quantitative growth and enzymatic activity assays, and used as patient avatars for PMM2-CDG drug screens yielding compounds that could be rapidly cross-validated in zebrafish, rodent and human organoid models.Phosphomannomutase 2 Deficiency (PMM2-CDG) is the most common monogenic congenital disorder of glycosylation (CDG) affecting at least 800 patients globally. PMM2 orthologs are present in model organisms, including the budding yeast  ALG1 (ALG12 (ALG2 (ALG3 (ALG8 (ALG9 (PGM3 (Phosphomannomutase 2 deficiency (PMM2-CDG) is the most common congenital disorder of glycosylation  complementation group in forward genetics screens in the early 1980s. These studies culminated three decades later in the 2013 Nobel Prize in Medicine and Physiology (SEC53 isomerize mannose-6-phosphate (M6P) to mannose-1-phosphate (M1P), and are activated by glucose-1,6-bisphosphate (G16) and mannose-1,6-bisphosphate (M16)  . The oliin vitro using recombinant enzyme expressed in bacteria and in patient-cell-derived assays. PMM2 protein variants were expressed in E. coli and displayed enzymatic activities between 0% and 100% of control levels, with most missense alleles exhibiting activity between 16–54% of control levels  or GenScript . Strains were generated by Next Interactions or in-house.All strains used in this study are in S288c background . Strains were grown in SC or SC drop out media (Sunrise Science) + 2% dextrose at 30° unless otherwise noted. Standard genetic procedures of transformation and tetrad analysis were followed to construct strains. 600 = 1.0, then serial diluted into 50 µL SC+FOA media in 384-well plates at 10−1, 10−2, and 10−3. Plates were incubated at 30° and absorbance reading at 600 nm were measured by a plate reader (Molecular Devices SpectraMax M3) at the indicated time points. Plates were vortexed briefly to resuspend cells prior to plate readings. 5-floroortic acid was purchased from US Biological and used at a concentration of 1 mg/L.Cells were picked from plates and resuspended in SC media to OD600’s worth of cells. Cells are washed once in 25 mM KPO4 pH 8.0 and resuspended in 600 µL lysis buffer  and an equivalent amount of glass beads. The bead/lysate mixture was vortexed for 7-10 cycles of 2 min vortex and 1 min cooling on ice. Lysis was checked by microscopy to confirm that 80–95% of cells are lysed. The lysates were clarified by 15 min centrifuge at max speed at 4° and the supernatant moved to a new tube. For long term storage at -80°, glycerol was added to 20% of the total volume. Protein concentration was determined with the Qubit protein assay kit (Thermo Fisher Scientific).Yeast lysates were prepared from 50 OD+ to NADPH in a reaction mixture incubated at 30° for 120 min in a 96-well plate. Cell lysates were added to a final concentration of 0.1 µg/mL protein in 200 µL volume in the following reaction: 50 mM HEPES, pH 7.1, 5 mM MgCl2, 0.5 mM NADP+, 10 µg/mL yeast glucose-6-phosphate dehydrogenase (Sigma G6378), 20 µM glucose 1,6-bisphosphate (Sigma 49225), 200 µM mannose 1-phosphate (Sigma M1755), 10 µg/mL phosphoglucose isomerase (Sigma P5381), and 5 µg/mL phosphomannose isomerase (Sigma P2621). Glucose 1,6-bisphosphate was used as an activator of PMM2 activity. NADPH formation was calculated from absorbance at 340 nm using the Beer’s Law (absorbance = εLc).PMM2 enzymatic activities were assayed spectrophotometrically at 340 nm by the reduction of NADP−2 dilution of an OD600 = 1.0 or 0.5 yeast cell suspensions were dispensed into the 384-well plates containing compounds or DMSO with a MultiFlo automated dispenser (Biotek). Plates were covered and incubated at 30° for 16 – 21 hr until OD600 reaches ∼0.8. Plates were vortexed briefly to resuspend cells prior to reading.125 nL of compounds or DMSO were dispensed into 384-well plates using the Echo acoustic dispenser (LabCyte) to achieve a final concentration of 25 µM. 50 µL of 10600 reaches ∼0.8. Plates were vortexed briefly to resuspend cells prior to plate readings.Microsource Spectrum compounds were reordered from MicroSource Discovery Systems and resuspended in DMSO. 5 – 125 nL compounds or DMSO were dispensed into 384-well plates and 50 µL of yeast cell suspensions or media were dispensed by multichannel pipettes to achieve the desired concentration. Plates were covered and incubated at 30° for 16 – 23 hr until ODhttps://doi.org/10.25387/g3.7408367 and consists of a list of strains and plasmids (Table S1) and five figures.Supplemental material available at Figshare: SEC53 are orthologs that share 55% identity at the amino-acid level   . Based oSEC53 is an essential gene, we placed a wildtype SEC53 copy on a URA3 plasmid that we can conditionally remove by growing cells in 5-fluoroorotic acid (5-FOA). 5-FOA is an analog of uracil that is converted into a toxic intermediate in cells where the uracil biosynthetic pathway is active, which the URA3 marker enables. Each SEC53 variant is then individually integrated at the HO locus of sec53∆ cells. The phenotype of each variant is revealed when the wildtype URA3 containing plasmid is counter-selected in media containing 5-FOA.To overcome the complication that sec53∆ negative control , and also grows better when its expression is doubled (57%). The relative growth of F126L and V238M is consistent with their reported residual in vitro enzymatic activity (TEF1 promoter completely rescues these cells. E100K is viable only under the ACT1 (16%) and TEF1 (67%) promoters, which indicates the severity of this mutation compared to the other variants. On the other hand, the R148H null allele is not compatible with growth at any promoter strength and phenocopies the genetic null sec53∆. Together, these data can be fully explained by mass action effects, where a reduction in enzymatic activity can be overcome by increasing the total amount of enzyme in the cell.Increasing the expression level of the hypomorphic alleles improves their growth in every case except the R148H variant and the SEC53 mRNA expression to 20% of its native level with the REV1 promoter modestly, but consistently, compromises cell growth to 64% of the control . This result suggests that the splicing defect of the 415G > A mutation in humans may reduce the abundance of still functional PMM2 E139K proteins to a sub-critical amount below the threshold for viability.In contrast, reducing wildtype  control . This reSEC53 alleles correlates with the level of residual enzymatic activity of the PMM2 alleles reported using bacterial recombinant protein and PMM2-CDG patient fibroblasts  paired with the R141H null allele. The most common genotype is PMM28H cells .600 = 0.93 ± 0.01) was growing better than the pSEC53-V238M/R148H heterozygous diploid (OD600 = 0.79 ± 0.2). This could be explained by the excess of R148H monomers relative to V238M monomers resulting in formation of nonfunctional R148H:R148H homodimers at a higher rate than hemi-functional V238M:R148H heterodimers.Homozygous F126L diploids grow slower than their respective F126L/R148H heterozygous diploids . In bothTEF1 promoter, E100K/E100K is 53% compared to 99% in E100K/WT and 100% in E100K/R148H. Under the ACT1 promoter, E100K/E100K is 8% compared to 100% in E100K/WT and 78% in E100K/R148H. Like F126, E100 is expected to affect dimerization and R148H may facilitate the formation of partially functional heterodimers. E146K, which is indistinguishable from wildtype, grew as expected except with the REV1 promoter: pREV1-E146K/E146K homozygous diploids (96%) grow better compared to the E146K/R148H heterozygous diploids (66%)  . One expSEC53, sec53-6  grows similarly to wildtype PMM2, in agreement with yeast SEC53 E146K. V231M compromises growth (56%) and F119L further compromises growth (45%). E93A, on the other hand, is not sufficient for growth under the SEC53 promoter and does not restore growth above sec53∆ cells. This is consistent with pSEC53-E100K and further supports the essentiality of E93 in PMM2 function.It was previously demonstrated that expression of human PMM2 rescues the lethality of a temperature-sensitive allele of st SEC53 . The degACT1 promoter improves growth to 97% . NADPH production is achieved by a series of four enzyme-coupled reactions shown in Supplemental Figure 4A. Mannose-1-phosphate and NADP+ are provided as substrates for the reactions along with phosphomannose isomerase, phosphoglucose isomerase, glucose-6-phosphoate dehydrogenase, and PMM2 from the protein lysate. Additionally, glucose 1,6-bisphosphate is used as an activator of PMM2 activity.We performed a PMM2 enzymatic assay to determine whether the expression level of the variants correlates with the enzymatic activity measured in wildtype and mutant yeast cell lysates. We assayed phosphomannomutase (PMM) activity by measuring the reduction of NADP+ to NADPH spectrophotometrically at 340 nm with a plate reader  and 24 wells of the positive controls (wildtype cells). As shown in We reordered these compounds from the vendor as dry powder to retest them in multi-point dose response assays. We found that three of the six compounds showed consistent and dose-dependent rescues in haploid and heterozygous diploid cells, as defined by an improvement of greater than 10% in growth . At the 2’-2’-bisepigallocatechin digallate rescues growth of pSEC53-F126L and pSEC53-V238M cells and exerts a stronger response in heterozygous diploid cells than haploids . Growth Similarly, suramin hexasodium also rescues growth of the pSEC53 variants more than the pACT1 variants, and diploids more strongly than haploids . At 100 In summary, we found that alpha-cyano-4-hydroxycinnamic acid specifically rescues pACT1-F126L cells, while 2’-2’-bisepigallocatechin digallate and suramin hexasodium preferentially rescue pSEC53-F126L/R148H and pSEC53-V238M/R148H heterozygous diploid cells.sec53∆ cells. Second, each of the five pathogenic PMM2 missense mutations studied herein and their orthologous SEC53 mutations support comparable yeast cell growth in a sec53∆ whole-gene deletion background. The rank-ordered severity of the mutation pairs from weakest to strongest loss-of-function is: E139K & E146K >> V231M & V238M > F119L & F126L >> E93A & E100K >> R141H & R148H. Third, the residual enzymatic activities reported in the literature for the five PMM2 variants are correlated to the extent of yeast cell growth rescued by these variants. Fourth, a pilot drug repurposing screen successfully identified three compounds – an FDA approved drug, a tool compound and a GRAS  plant-based natural product.We created new yeast models of the congenital disorder of glycosylation PMM2-CDG and demonstrated their relevance toward developing therapeutics in four ways. First, yeast-codon-optimized human PMM2 fully rescues the lethality of REV1 promoter, or 20% of the native SEC53 promoter strength. The two dimerization-defective variants F126L and E100K grew better when in trans with R148H than as homozygotes because R148H monomers can dimerize with either F126L or E100K monomers to form hemi-functional heterodimers. On the other hand, the known strongly folding-defective variant V238M and the weakly hypomorphic variant E146K grew better in homozygosity than in trans with R148H. We speculate that a larger pool of R148H monomers relative to V238M or E146K monomers due to the greater stability of the former results in a higher proportion of nonfunctional R148H:R148H homodimers diluting the total pool of fully functional V238M or E146K homodimers. Collectively, these results suggest two different therapeutic approaches, one for when a PMM2 allele is dimerization defective and another for when a PMM2 allele is folding defective or prone to aggregation and rapid turnover.The three most well studied variants – R141H, F119L and V231M – all behaved as expected in haploid and homozygous diploid cells. A residual PMM2 enzymatic activity is not reported for bacterially expressed recombinant E93A protein, and our results clearly indicate the strong loss-of-function of both E93A and E100K variants in haploid and homozygous diploid cells. Contrary to the reported 25% residual PMM2 enzymatic activity of the E139K variant, our results indicate that both E139K and E146K variants grow similarly to wildtype controls with only a modest growth defect evident at the lowest expressing F119L/R141H patient fibroblast cells show 10% enzymatic activity. We infer from the severe growth defects of SEC53 E100K strains that the residual enzymatic activity of the E93A variant must be less than that of F119L and below 7%.Based on the results of the phosphomannomutase enzymatic assay we adapted for yeast cell lysates, we challenge the reported residual enzymatic activities of F119L and V231M as 25% and 37.5% of control levels, which were determined using bacterially expressed recombinant PMM2 variants. Our data showed lower residual phosphomannomutase activity with V238M at ∼10% of control levels and that of F119L at ∼7% of control levels. In our hands, PMM2The pharmacology of the three hits from the Microsource Spectrum screen suggests possible explanations for rescue of PMM2-CDG involving antioxidant effects and perturbation of glycolysis. alpha-cyano-4-hydroxycinnamic acid has been described as a monocarboxylate transporter inhibitor (These yeast based PMM2-CDG patient avatars not only can be used to identify potential therapeutics demonstrated in this study, but can also be used in genetic modifier screens, genetic interaction screens, and basic studies to elucidate the pathophysiological cascade of PMM2-CDG starting at the cellular level and working up in model organism complexity.