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1.
Nat Commun ; 13(1): 5435, 2022 09 16.
Article in English | MEDLINE | ID: mdl-36114200

ABSTRACT

Covalent attachment of ubiquitin (Ub) to proteins is a highly versatile posttranslational modification. Moreover, Ub is not only a modifier but itself is modified by phosphorylation and lysine acetylation. However, the functional consequences of Ub acetylation are poorly understood. By generation and comprehensive characterization of all seven possible mono-acetylated Ub variants, we show that each acetylation site has a particular impact on Ub structure. This is reflected in selective usage of the acetylated variants by different E3 ligases and overlapping but distinct interactomes, linking different acetylated variants to different cellular pathways. Notably, not only electrostatic but also steric effects contribute to acetylation-induced changes in Ub structure and, thus, function. Finally, we provide evidence that p300 acts as a position-specific Ub acetyltransferase and HDAC6 as a general Ub deacetylase. Our findings provide intimate insights into the structural and functional consequences of Ub acetylation and highlight the general importance of Ub acetylation.


Subject(s)
Lysine , Ubiquitin , Acetylation , Acetyltransferases/metabolism , Lysine/metabolism , Protein Processing, Post-Translational , Static Electricity , Ubiquitin/metabolism , Ubiquitin-Protein Ligases/metabolism
2.
Nat Commun ; 12(1): 5939, 2021 10 12.
Article in English | MEDLINE | ID: mdl-34642328

ABSTRACT

Ubiquitin (Ub) and Ub-like proteins (Ubls) such as NEDD8 are best known for their function as covalent modifiers of other proteins but they are also themselves subject to post-translational modifications including phosphorylation. While functions of phosphorylated Ub (pUb) have been characterized, the consequences of Ubl phosphorylation remain unclear. Here we report that NEDD8 can be phosphorylated at S65 - the same site as Ub - and that S65 phosphorylation affects the structural dynamics of NEDD8 and Ub in a similar manner. While both pUb and phosphorylated NEDD8 (pNEDD8) can allosterically activate the Ub ligase Parkin, they have different protein interactomes that in turn are distinct from those of unmodified Ub and NEDD8. Among the preferential pNEDD8 interactors are HSP70 family members and we show that pNEDD8 stimulates HSP70 ATPase activity more pronouncedly than unmodified NEDD8. Our findings highlight the general importance of Ub/NEDD8 phosphorylation and support the notion that the function of pUb/pNEDD8 does not require their covalent attachment to other proteins.


Subject(s)
HSP70 Heat-Shock Proteins/metabolism , NEDD8 Protein/metabolism , Protein Processing, Post-Translational , Ubiquitin/metabolism , Allosteric Regulation , Amino Acid Sequence , Cloning, Molecular , Escherichia coli/genetics , Escherichia coli/metabolism , Gene Expression , Genetic Vectors/chemistry , Genetic Vectors/metabolism , HEK293 Cells , HSP70 Heat-Shock Proteins/chemistry , HSP70 Heat-Shock Proteins/classification , HSP70 Heat-Shock Proteins/genetics , Humans , Kinetics , NEDD8 Protein/chemistry , NEDD8 Protein/genetics , Phosphorylation , Protein Binding , Protein Interaction Mapping , Recombinant Proteins/chemistry , Recombinant Proteins/genetics , Recombinant Proteins/metabolism , Sequence Alignment , Sequence Homology, Amino Acid , Substrate Specificity , Ubiquitin/chemistry , Ubiquitin/genetics , Ubiquitination
3.
Nat Commun ; 12(1): 5808, 2021 10 04.
Article in English | MEDLINE | ID: mdl-34608152

ABSTRACT

The nucleotides diadenosine triphosphate (Ap3A) and diadenosine tetraphosphate (Ap4A) are formed in prokaryotic and eukaryotic cells. Since their concentrations increase significantly upon cellular stress, they are considered to be alarmones triggering stress adaptive processes. However, their cellular roles remain elusive. To elucidate the proteome-wide interactome of Ap3A and Ap4A and thereby gain insights into their cellular roles, we herein report the development of photoaffinity-labeling probes and their employment in chemical proteomics. We demonstrate that the identified ApnA interactors are involved in many fundamental cellular processes including carboxylic acid and nucleotide metabolism, gene expression, various regulatory processes and cellular response mechanisms and only around half of them are known nucleotide interactors. Our results highlight common functions of these ApnAs across the domains of life, but also identify those that are different for Ap3A or Ap4A. This study provides a rich source for further functional studies of these nucleotides and depicts useful tools for characterization of their regulatory mechanisms in cells.


Subject(s)
Dinucleoside Phosphates/metabolism , Proteomics , Adenosine Triphosphate/metabolism , Dinucleoside Phosphates/chemistry , Endoribonucleases/metabolism , Escherichia coli/metabolism , Escherichia coli Proteins/metabolism , HEK293 Cells , Humans , L-Lactate Dehydrogenase/metabolism , Phosphoglycerate Kinase/metabolism , Photoaffinity Labels/chemical synthesis , Photoaffinity Labels/chemistry , Photoaffinity Labels/metabolism , Protein Binding , Ubiquitin-Activating Enzymes/metabolism
4.
Chemistry ; 26(28): 6279-6284, 2020 May 15.
Article in English | MEDLINE | ID: mdl-32154932

ABSTRACT

Simple and robust assays to monitor enzymatic ATP cleavage with high efficiency in real-time are scarce. To address this shortcoming, we developed fluorescently labelled adenosine tri-, tetra- and pentaphosphate analogues of ATP. The novel ATP analogues bear - in contrast to earlier reports - only a single acridone-based dye at the terminal phosphate group. The dye's fluorescence is quenched by the adenine component of the ATP analogue and is restored upon cleavage of the phosphate chain and dissociation of the dye from the adenosine moiety. Thereby the activity of ATP-cleaving enzymes can be followed in real-time. We demonstrate this proficiency for ubiquitin activation by the ubiquitin-activating enzymes UBA1 and UBA6 which represents the first step in an enzymatic cascade leading to the covalent attachment of ubiquitin to substrate proteins, a process that is highly conserved from yeast to humans. We found that the efficiency to serve as cofactor for UBA1/UBA6 very much depends on the length of the phosphate chain of the ATP analogue: triphosphates are used poorly while pentaphosphates are most efficiently processed. Notably, the novel pentaphosphate-harbouring ATP analogue supersedes the efficiency of recently reported dual-dye labelled analogues and thus, is a promising candidate for broad applications.


Subject(s)
Adenosine Triphosphate/chemistry , Fluorescent Dyes/chemistry , Ubiquitin-Activating Enzymes/metabolism , Adenosine Triphosphate/analogs & derivatives , Adenosine Triphosphate/metabolism , Fluorescent Dyes/metabolism , Humans , Ubiquitin-Activating Enzymes/chemistry
5.
Cell Chem Biol ; 26(11): 1535-1543.e5, 2019 Nov 21.
Article in English | MEDLINE | ID: mdl-31492597

ABSTRACT

Diadenosine polyphosphates (ApnAs) such as diadenosine tri- and tetraphosphates are formed in prokaryotic as well as eukaryotic cells. Since upon stress intracellular ApnA concentrations increase, it was postulated that ApnAs are alarmones triggering stress-adaptive processes. The major synthesis pathway of ApnAs is assumed to be a side reaction of amino acid activation. How this process is linked to stress adaptation remains enigmatic. The first step of one of the most prominent eukaryotic post-translational modification systems-the conjugation of ubiquitin (Ub) and ubiquitin-like proteins (Ubl) to target proteins-involves the formation of an adenylate as intermediate. Like ApnA formation, Ub and Ubl conjugation is significantly enhanced during stress conditions. Here, we demonstrate that diadenosine tri- and tetraphosphates are indeed synthesized during activation of Ub and Ubls. This links one of the most prevalent eukaryotic protein-modification systems to ApnA formation for the first time.


Subject(s)
Dinucleoside Phosphates/metabolism , Ubiquitin-Activating Enzymes/metabolism , Ubiquitin/metabolism , Biocatalysis , Chromatography, High Pressure Liquid , Dinucleoside Phosphates/analysis , Humans , Mass Spectrometry , Mutagenesis , Recombinant Proteins/biosynthesis , Small Ubiquitin-Related Modifier Proteins/genetics , Small Ubiquitin-Related Modifier Proteins/metabolism , Ubiquitin/genetics , Ubiquitin-Activating Enzymes/genetics
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