Crystal Structure of the N-Acetylmannosamine Kinase Domain of GNE

UDP-GlcNAc 2-epimerase/ManNAc 6-kinase, GNE, is a bi-functional enzyme that plays a key role in sialic acid biosynthesis. Mutations of the GNE protein cause sialurea or autosomal recessive inclusion body myopathy/Nonaka myopathy. GNE is the only human protein that contains a kinase domain belonging to the ROK (repressor, ORF, kinase) family.We solved the structure of the GNE kinase domain in the ligand-free state. The protein exists predominantly as a dimer in solution, with small populations of monomer and higher-order oligomer in equilibrium with the dimer. Crystal packing analysis reveals the existence of a crystallographic hexamer, and that the kinase domain dimerizes through the C-lobe subdomain. Mapping of disease-related missense mutations onto the kinase domain structure revealed that the mutation sites could be classified into four different groups based on the location - dimer interface, interlobar helices, protein surface, or within other secondary structural elements.The crystal structure of the kinase domain of GNE provides a structural basis for understanding disease-causing mutations and a model of hexameric wild type full length enzyme.This article can also be viewed as an enhanced version in which the text of the article is integrated with interactive 3D representations and animated transitions. Please note that a web plugin is required to access this enhanced functionality. Instructions for the installation and use of the web plugin are available in Text S1

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

Colloidal Aggregation and the in Vitro Activity of Traditional Chinese Medicines

Traditional Chinese Medicines (TCMs) have been the sole source of therapeutics in China for two millennia. In recent drug discovery efforts, purified components of TCM formulations have shown activity in many in vitro assays, raising concerns of promiscuity. Here, we investigated 14 bioactive small molecules isolated from TCMs for colloidal aggregation. At concentrations commonly used in cell-based or biochemical assay conditions, eight of these compounds formed particles detectable by dynamic light scattering and showed detergent-reversible inhibition against β-lactamase and malate dehydrogenase, two counter-screening enzymes. When three of these compounds were tested against their literature-reported molecular targets, they showed similar reversal of their inhibitory activity in the presence of detergent. For three of the most potent aggregators, contributions to promiscuity via oxidative cycling were investigated; addition of 1 mM DTT had no effect on their activity, which is inconsistent with an oxidative mechanism. TCMs are often active at micromolar concentrations; this study suggests that care must be taken to control for artifactual activity when seeking their primary targets. Implications for the formulation of these molecules are considered

Colloidal drug formulations can explain "bell-shaped" concentration-response curves

Drug efficacy does not always increase sigmoidally with concentration, which has puzzled the community for decades. Unlike standard sigmoidal curves, bell-shaped concentration-response curves suggest more complex biological effects, such as multiple-binding sites or multiple targets. Here, we investigate a physical property-based mechanism for bell-shaped curves. Beginning with the observation that some drugs form colloidal aggregates at relevant concentrations, we determined concentration-response curves for three aggregating anticancer drugs, formulated both as colloids and as free monomer. Colloidal formulations exhibited bell-shaped curves, losing activity at higher concentrations, while monomeric formulations gave typical sigmoidal curves, sustaining a plateau of maximum activity. Inverting the question, we next asked if molecules with bell-shaped curves, reported in the literature, form colloidal aggregates at relevant concentrations. We selected 12 molecules reported to have bell-shaped concentration-response curves and found that five of these formed colloids. To understand the mechanism behind the loss of activity at concentrations where colloids are present, we investigated the diffusion of colloid-forming dye Evans blue into cells. We found that colloidal species are excluded from cells, which may explain the mechanism behind toxicological screens that use Evans blue, Trypan blue, and related dyes

Nicotinamide Riboside Kinase Structures Reveal New Pathways to NAD þ

The eukaryotic nicotinamide riboside kinase (Nrk) pathway, which is induced in response to nerve damage and promotes replicative life span in yeast, converts nicotinamide riboside to nicotinamide adenine dinucleotide (NAD þ)by phosphorylation and adenylylation. Crystal structures of human Nrk1 bound to nucleoside and nucleotide substrates and products revealed an enzyme structurally similar to Rossmann fold metabolite kinases and allowed the identification of active site residues, which were shown to be essential for human Nrk1 and Nrk2 activity in vivo. Although the structures account for the 500-fold discrimination between nicotinamide riboside and pyrimidine nucleosides, no enzyme feature was identified to recognize the distinctive carboxamide group of nicotinamide riboside. Indeed, nicotinic acid riboside is a specific substrate of human Nrk enzymes and is utilized in yeast in a novel biosynthetic pathway that depends on Nrk and NAD þ synthetase. Additionally, nicotinic acid riboside is utilized in vivo by Urh1, Pnp1, and Preiss-Handler salvage. Thus, crystal structures of Nrk1 led to the identification of new pathways to NAD þ

Virtual Screening for UDP-Galactopyranose Mutase Ligands Identifies a New Class of Antimycobacterial Agents

Galactofuranose (Galf) is present in glycans critical for the virulence and viability of several pathogenic microbes, including Mycobacterium tuberculosis, yet the monosaccharide is absent from mammalian glycans. Uridine 5′-diphosphate-galactopyranose mutase (UGM) catalyzes the formation of UDP-Galf, which is required to produce Galf-containing glycoconjugates. Inhibitors of UGM have therefore been sought, both as antimicrobial leads and as tools to delineate the roles of Galf in cells. Obtaining cell permeable UGM probes by either design or high throughput screens has been difficult, as has elucidating how UGM binds small molecule, noncarbohydrate inhibitors. To address these issues, we employed structure-based virtual screening to uncover new inhibitor chemotypes, including a triazolothiadiazine series. These compounds are among the most potent antimycobacterial UGM inhibitors described. They also facilitated determination of a UGM–small molecule inhibitor structure, which can guide optimization. A comparison of results from the computational screen and a high-throughput fluorescence polarization (FP) screen indicated that the scaffold hits from the former had been evaluated in the FP screen but missed. By focusing on promising compounds, the virtual screen rescued false negatives, providing a blueprint for generating new UGM probes and therapeutic leads. [Image: see text

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)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

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