N-terminal acetylation shields proteins from degradation and promotes age-dependent motility and longevity

Most eukaryotic proteins are N-terminally acetylated, but the functional impact on a global scale has remained obscure. Using genome-wide CRISPR knockout screens in human cells, we reveal a strong genetic dependency between a major N-terminal acetyltransferase and specific ubiquitin ligases. Biochemical analyses uncover that both the ubiquitin ligase complex UBR4-KCMF1 and the acetyltransferase NatC recognize proteins bearing an unacetylated N-terminal methionine followed by a hydrophobic residue. NatC KO-induced protein degradation and phenotypes are reversed by UBR knockdown, demonstrating the central cellular role of this interplay. We reveal that loss of Drosophila NatC is associated with male sterility, reduced longevity, and age-dependent loss of motility due to developmental muscle defects. Remarkably, muscle-specific overexpression of UbcE2M, one of the proteins targeted for NatC KO-mediated degradation, suppresses defects of NatC deletion. In conclusion, NatC-mediated N-terminal acetylation acts as a protective mechanism against protein degradation, which is relevant for increased longevity and motility. The most common protein modification in eukaryotes is N-terminal acetylation, but its functional impact has remained enigmatic. Here, the authors find that a key role for N-terminal acetylation is shielding proteins from ubiquitin ligase-mediated degradation, mediating motility and longevity.Association Francaise contre les Myopathies 261981, Canadian Institutes of Health Research (CIHR) 249843, United States Department of Health & Human Services National Institutes of Health (NIH) - USA F-12540, Portuguese national funding through Fundaco para a Ciencia e a Tecnologia (FCT) 171752-PR-2009-0222, National Funds through Fundaco para a Ciencia e a Tecnologia (FCT) G008018N, G002721N, University of Bergen MC_UU_00028/6, FDN-143264, FDN-143265, PJT-180285, PJT-463531, R01HG005853, R01HG005084, DL 57/2016/CP1361/CT0019, 2022.01782.PTDC,PTDC/BIA-BID/28441/2017,PTDC/BIA-BID/1606/2020, ALG-01-0145-FEDER-028441, PPBI-POCI-01-0145-FEDER-022122, LISBOA-01-0145-FEDER-022170info:eu-repo/semantics/publishedVersio

N-terminal modifications of cellular proteins: the enzymes involved, their substrate specificities and biological effects

The vast majority of eukaryotic proteins are N-terminally modified by one or more processing enzymes. Enzymes acting on the very first amino acid of a polypeptide include different peptidases, transferases, and ligases. Methionine aminopeptidases excise the initiator methionine leaving the nascent polypeptide with a newly exposed amino acid that may be further modified. N-terminal acetyl-, methyl-, myristoyl-, and palmitoyltransferases may attach an acetyl, methyl, myristoyl, or palmitoyl group, respectively, to the α-amino group of the target protein N-terminus. With the action of ubiquitin ligases, one or several ubiquitin molecules are transferred, and hence, constitute the N-terminal modification. Modifications at protein N-termini represent an important contribution to proteomic diversity and complexity, and are essential for protein regulation and cellular signaling. Consequently, dysregulation of the N-terminal modifying enzymes is implicated in human diseases. We here review the different protein N-terminal modifications occurring co- or post-translationally with emphasis on the responsible enzymes and their substrate specificities

N-Terminal Acetylation by NatC Is Not a General Determinant for Substrate Subcellular Localization in <i>Saccharomyces cerevisiae</i>

<div><p>N-terminal acetylation has been suggested to play a role in the subcellular targeting of proteins, in particular those acetylated by the N-terminal acetyltransferase complex NatC. Based on previous positional proteomics data revealing N-terminal acetylation status and the predicted NAT substrate classes, we selected 13 suitable NatC substrates for subcellular localization studies in <i>Saccharomyces cerevisiae</i>. Fluorescence microscopy analysis of GFP-tagged candidates in the presence or absence of the NatC catalytic subunit Naa30 (Mak3) revealed unaltered localization patterns for all 13 candidates, thus arguing against a general role for the N-terminal acetyl group as a localization determinant. Furthermore, all organelle-localized substrates indicated undisrupted structures, thus suggesting that absence of NatC acetylation does not have a vast effect on organelle morphology in yeast.</p></div

NatC substrates included in this study.

1<p>As described by Huh <i>et al</i>. <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0061012#pone.0061012-Lai1" target="_blank">[23]</a> and observed in this study.</p

Subcellular localizations of putative NatC substrates in wild type and <i>naa30</i>Δ cells.

<p>GFP-localization patterns for all 13 candidates were unaffected by <i>NAA30</i>-deletion. The Golgi localized Sec18; ER localized Sly41; nuclear membrane localized Nup157; nucleolus localized Rrn11; nucleus localized Rfc2; mitochondrial Ymr31 and Pda1; cytosolic and nuclear Glr1; cytosolic Tma20; bud neck localized Lrg1, Bem1 and Pxl1; and a protein localized to lipid particle membranes, Tgl1, were all unaffected in their subcellular localization pattern as investigated by fluorescence microscopy. GFP-fusion proteins are endogenously expressed. Scale bar 2 µm.</p

Subcellular localization of Arl3 and Trm1 in wild type and <i>naa30</i>Δ cells.

<p>The defined subcellular localization of Arl3 and Trm1-II was lost in <i>naa30</i>Δ cells. Arl3 and Trm1-GFP-fusion proteins are endogenously expressed. Trm1-GFP, gives rise to both forms (I and II) of Trm1, resulting from alternative translation initiation sites. See text for details. Scale bar 2 µm.</p

Expanded in vivo substrate profile of the yeast N-terminal acetyltransferase NatC

N-terminal acetylation is a conserved protein modification among eukaryotes. The yeast Saccharomyces cerevisiae is a valuable model system for studying this modification. The bulk of protein N-terminal acetylation in S. cerevisiae is catalyzed by the N-terminal acetyltransferases NatA, NatB, and NatC. Thus far, proteome-wide identification of the in vivo protein substrates of yeast NatA and NatB has been performed by N-terminomics. Here, we used S. cerevisiae deleted for the NatC catalytic subunit Naa30 and identified 57 yeast NatC substrates by N-terminal combined fractional diagonal chromatography analysis. Interestingly, in addition to the canonical N-termini starting with ML, MI, MF, and MW, yeast NatC substrates also included MY, MK, MM, MA, MV, and MS. However, for some of these substrate types, such as MY, MK, MV, and MS, we also uncovered (residual) non-NatC NAT activity, most likely due to the previously established redundancy between yeast NatC and NatE/Naa50. Thus, we have revealed a complex interplay between different NATs in targeting methionine-starting N-termini in yeast. Furthermore, our results showed that ectopic expression of human NAA30 rescued known NatC phenotypes in naa30Δ yeast, as well as partially restored the yeast NatC Nt-acetylome. Thus, we demonstrate an evolutionary conservation of NatC from yeast to human thereby underpinning future disease models to study pathogenic NAA30 variants. Overall, this work offers increased biochemical and functional insights into NatC-mediated N-terminal acetylation and provides a basis for future work to pinpoint the specific molecular mechanisms that link the lack of NatC-mediated N-terminal acetylation to phenotypes of NatC deletion yeast.publishedVersio

N-terminal acetylation shields proteins from degradation and promotes age-dependent motility and longevity

International audienceSUMMARY Most eukaryotic proteins are N-terminally acetylated, but the functional impact on a global scale has remained obscure. Using genome-wide CRISPR knockout screens in human cells, we reveal a strong genetic dependency between a major N-terminal acetyltransferase and specific ubiquitin ligases. Biochemical analyses uncover that both the ubiquitin ligase complex UBR4-KCMF1 and the acetyltransferase NatC recognize proteins bearing an unacetylated N-terminal methionine followed by a hydrophobic residue. NatC KO-induced protein degradation and phenotypes are reversed by UBR knockdown, demonstrating the central cellular role of this interplay. We reveal that loss of Drosophila NatC is associated with male sterility, reduced longevity, and age-dependent loss of motility due to developmental muscle defects. Remarkably, muscle-specific overexpression of UbcE2M, one of the proteins targeted for NatC KO mediated degradation, suppresses defects of NatC deletion. In conclusion, NatC-mediated N-terminal acetylation acts as a protective mechanism against protein degradation, which is relevant for increased longevity and motility. In Brief Varland, Silva et al . define that a major cellular role of N-terminal acetylation is shielding proteins from proteasomal degradation by specific ubiquitin ligases. The human N-terminal acetyltransferase NatC protects the neddylation regulator UBE2M from degradation, while overexpression of Drosophila UBE2M/UbcE2M rescues the longevity and motility defects of NatC deletion. Highlights N-terminal acetylation by NatC protects proteins from degradation, including UBE2M UBR4-KCMF1 targets unacetylated N-terminal Met followed by a hydrophobic residue Drosophila NatC is required for adult longevity and motility in elderly Overexpression of UBE2M/UbcE2M suppresses Drosophila NatC deletion phenotype

N-terminal acetylation shields proteins from degradation and promotes age-dependent motility and longevity

Acknowledgements: We thank members of the Arnesen, Martinho, Boone, and Andrews labs for helpful discussions. Lena Thurnes, Kjellfrid Haukenes, Nina Glomnes, Olga Sizova, and Andrea Habsid are gratefully acknowledged for technical assistance. We also thank Dr. Jaakko Saraste and Dr. Marc Niere for help and advice. Furthermore, we thank Dr. Scott J. Dixon and Dr. Stian Knappskog for cell lines, Dr. Eileen Furlong for antibody, as well as Dr. Yong Tae Kwon, Dr. Fabio Demontis, and Dr. Liam C. Hunt for kindly providing plasmids. We also thank Dr. Paulo Navarro-Costa for his contribution to the Drosophila testis analysis. Next-generations sequencing was performed at the Centre for Applied Genomics, The Hospital for Sick Children, Toronto, Canada. Proteomics work of human cells lines was performed at the VIB Proteomics Core, located at the VIB-UGent Center for Medical Biotechnology, Belgium, and was supported by EPIC-XS, project number 823839, funded by the Horizon 2020 programme of the European Union. Proteomics work of Drosophila purified proteins was performed at the Mass Spectrometry Laboratory, Institute of Biochemistry and Biophysics, Polish Academy of Sciences, Warsaw, Poland. Sequencing was performed at Haukeland University Hospital. Flow cytometry analysis and cell sorting were performed at the Flow Cytometry Core Facility, Department of Clinical Science, University of Bergen, Norway, with advice from Dr. Brith Bergum. Confocal imaging was performed at the Molecular Imaging Center (MIC), Department of Biomedicine, University of Bergen. This work was supported by grants from the Research Council of Norway (RCN) (FRIPRO Mobility Grant 261981 to S.V. which was co-funded by the European Union’s Seventh Framework Programme under Marie Curie grant agreement no 608695, and FRIPRO grant 249843 to T.A.), the Norwegian Health Authorities of Western Norway (F-12540 to T.A.), the Norwegian Cancer Society (171752-PR-2009-0222 to T.A), the European Research Council (ERC) under the European Union Horizon 2020 Research and Innovation Program (Grant 772039 to T.A), the Research Foundation - Flanders (FWO) (G008018N and G002721N to K.G.), the French National Centre for Scientific Research (CNRS) (ATIP-Avenir grant to H.B), AFM-Telethon (Trampoline grant 23108 to H.B.), the Medical Research Council (MRC) (MC_UU_00028/6 to A.J.W), the Canadian Institutes of Health Research (CIHR) (FDN-143264 and FDN-143265 to C.B. and B.J.A.; PJT-180285 to C.B, PJT-463531 to J.M.), the National Institutes of Health (NIH) (R01HG005853 to B.J.A., C.B., and C.L.M. R01HG005084 to C.L.M.), and the Portuguese national funding through Fundação para a Ciência e a Tecnologia (FCT) (DL 57/2016/CP1361/CT0019 and 2022.01782.PTDC to R.D.S; PTDC/BIA-BID/28441/2017 and PTDC/BIA-BID/1606/2020 to R.G.M). This particular work was partially funded by Algarve Regional Operational Program - CRESC Algarve 2020 and by National Funds through Fundação para a Ciência e a Tecnologia (FCT), under the project ALG-01-0145-FEDER-028441. The Algarve Biomedical Center Research Institute Microscopy Unit was partially supported by the Portuguese national funding (PPBI-POCI-01-0145-FEDER-022122). This work was developed with the support of the research infrastructure Congento (LISBOA-01-0145-FEDER-022170). The funding bodies had no roles in study design, data collection, data analysis, data interpretation, or manuscript writing.Funder: U.S. Department of Health & Human Services | NIH | Office of Extramural Research, National Institutes of Health (OER)AbstractMost eukaryotic proteins are N-terminally acetylated, but the functional impact on a global scale has remained obscure. Using genome-wide CRISPR knockout screens in human cells, we reveal a strong genetic dependency between a major N-terminal acetyltransferase and specific ubiquitin ligases. Biochemical analyses uncover that both the ubiquitin ligase complex UBR4-KCMF1 and the acetyltransferase NatC recognize proteins bearing an unacetylated N-terminal methionine followed by a hydrophobic residue. NatC KO-induced protein degradation and phenotypes are reversed by UBR knockdown, demonstrating the central cellular role of this interplay. We reveal that loss of Drosophila NatC is associated with male sterility, reduced longevity, and age-dependent loss of motility due to developmental muscle defects. Remarkably, muscle-specific overexpression of UbcE2M, one of the proteins targeted for NatC KO-mediated degradation, suppresses defects of NatC deletion. In conclusion, NatC-mediated N-terminal acetylation acts as a protective mechanism against protein degradation, which is relevant for increased longevity and motility.</jats:p

N-terminal acetylation shields proteins from degradation and promotes age-dependent motility and longevity

Acknowledgements: We thank members of the Arnesen, Martinho, Boone, and Andrews labs for helpful discussions. Lena Thurnes, Kjellfrid Haukenes, Nina Glomnes, Olga Sizova, and Andrea Habsid are gratefully acknowledged for technical assistance. We also thank Dr. Jaakko Saraste and Dr. Marc Niere for help and advice. Furthermore, we thank Dr. Scott J. Dixon and Dr. Stian Knappskog for cell lines, Dr. Eileen Furlong for antibody, as well as Dr. Yong Tae Kwon, Dr. Fabio Demontis, and Dr. Liam C. Hunt for kindly providing plasmids. We also thank Dr. Paulo Navarro-Costa for his contribution to the Drosophila testis analysis. Next-generations sequencing was performed at the Centre for Applied Genomics, The Hospital for Sick Children, Toronto, Canada. Proteomics work of human cells lines was performed at the VIB Proteomics Core, located at the VIB-UGent Center for Medical Biotechnology, Belgium, and was supported by EPIC-XS, project number 823839, funded by the Horizon 2020 programme of the European Union. Proteomics work of Drosophila purified proteins was performed at the Mass Spectrometry Laboratory, Institute of Biochemistry and Biophysics, Polish Academy of Sciences, Warsaw, Poland. Sequencing was performed at Haukeland University Hospital. Flow cytometry analysis and cell sorting were performed at the Flow Cytometry Core Facility, Department of Clinical Science, University of Bergen, Norway, with advice from Dr. Brith Bergum. Confocal imaging was performed at the Molecular Imaging Center (MIC), Department of Biomedicine, University of Bergen. This work was supported by grants from the Research Council of Norway (RCN) (FRIPRO Mobility Grant 261981 to S.V. which was co-funded by the European Union’s Seventh Framework Programme under Marie Curie grant agreement no 608695, and FRIPRO grant 249843 to T.A.), the Norwegian Health Authorities of Western Norway (F-12540 to T.A.), the Norwegian Cancer Society (171752-PR-2009-0222 to T.A), the European Research Council (ERC) under the European Union Horizon 2020 Research and Innovation Program (Grant 772039 to T.A), the Research Foundation - Flanders (FWO) (G008018N and G002721N to K.G.), the French National Centre for Scientific Research (CNRS) (ATIP-Avenir grant to H.B), AFM-Telethon (Trampoline grant 23108 to H.B.), the Medical Research Council (MRC) (MC_UU_00028/6 to A.J.W), the Canadian Institutes of Health Research (CIHR) (FDN-143264 and FDN-143265 to C.B. and B.J.A.; PJT-180285 to C.B, PJT-463531 to J.M.), the National Institutes of Health (NIH) (R01HG005853 to B.J.A., C.B., and C.L.M. R01HG005084 to C.L.M.), and the Portuguese national funding through Fundação para a Ciência e a Tecnologia (FCT) (DL 57/2016/CP1361/CT0019 and 2022.01782.PTDC to R.D.S; PTDC/BIA-BID/28441/2017 and PTDC/BIA-BID/1606/2020 to R.G.M). This particular work was partially funded by Algarve Regional Operational Program - CRESC Algarve 2020 and by National Funds through Fundação para a Ciência e a Tecnologia (FCT), under the project ALG-01-0145-FEDER-028441. The Algarve Biomedical Center Research Institute Microscopy Unit was partially supported by the Portuguese national funding (PPBI-POCI-01-0145-FEDER-022122). This work was developed with the support of the research infrastructure Congento (LISBOA-01-0145-FEDER-022170). The funding bodies had no roles in study design, data collection, data analysis, data interpretation, or manuscript writing.Funder: U.S. Department of Health & Human Services | NIH | Office of Extramural Research, National Institutes of Health (OER)Most eukaryotic proteins are N-terminally acetylated, but the functional impact on a global scale has remained obscure. Using genome-wide CRISPR knockout screens in human cells, we reveal a strong genetic dependency between a major N-terminal acetyltransferase and specific ubiquitin ligases. Biochemical analyses uncover that both the ubiquitin ligase complex UBR4-KCMF1 and the acetyltransferase NatC recognize proteins bearing an unacetylated N-terminal methionine followed by a hydrophobic residue. NatC KO-induced protein degradation and phenotypes are reversed by UBR knockdown, demonstrating the central cellular role of this interplay. We reveal that loss of Drosophila NatC is associated with male sterility, reduced longevity, and age-dependent loss of motility due to developmental muscle defects. Remarkably, muscle-specific overexpression of UbcE2M, one of the proteins targeted for NatC KO-mediated degradation, suppresses defects of NatC deletion. In conclusion, NatC-mediated N-terminal acetylation acts as a protective mechanism against protein degradation, which is relevant for increased longevity and motility