Your browser doesn't support javascript.
loading
: 20 | 50 | 100
1 - 11 de 11
1.
Nat Commun ; 15(1): 1674, 2024 Feb 23.
Article En | MEDLINE | ID: mdl-38395951

The Escherichia coli TetR-related transcriptional regulator RutR is involved in the coordination of pyrimidine and purine metabolism. Here we report that lysine acetylation modulates RutR function. Applying the genetic code expansion concept, we produced site-specifically lysine-acetylated RutR proteins. The crystal structure of lysine-acetylated RutR reveals how acetylation switches off RutR-DNA-binding. We apply the genetic code expansion concept in E. coli in vivo revealing the consequences of RutR acetylation on the transcriptional level. We propose a model in which RutR acetylation follows different kinetic profiles either reacting non-enzymatically with acetyl-phosphate or enzymatically catalysed by the lysine acetyltransferases PatZ/YfiQ and YiaC. The NAD+-dependent sirtuin deacetylase CobB reverses enzymatic and non-enzymatic acetylation of RutR playing a dual regulatory and detoxifying role. By detecting cellular acetyl-CoA, NAD+ and acetyl-phosphate, bacteria apply lysine acetylation of transcriptional regulators to sense the cellular metabolic state directly adjusting gene expression to changing environmental conditions.


Escherichia coli Proteins , Escherichia coli , Escherichia coli/metabolism , Escherichia coli Proteins/metabolism , Lysine/metabolism , Acetylation , NAD/metabolism , Gene Expression , Phosphates/metabolism
2.
J Biol Chem ; 298(9): 102265, 2022 09.
Article En | MEDLINE | ID: mdl-35850304

Osteoporosis and multiple sclerosis are highly prevalent diseases with limited treatment options. In light of these unmet medical needs, novel therapeutic approaches are urgently sought. Previously, the activation of the transmembrane receptor Plexin-B1 by its ligand semaphorin 4D (Sema4D) has been shown to suppress bone formation and promote neuroinflammation in mice. However, it is unclear whether inhibition of this receptor-ligand interaction by an anti-Plexin-B1 antibody could represent a viable strategy against diseases related to these processes. Here, we raised and systematically characterized a monoclonal antibody directed against the extracellular domain of human Plexin-B1, which specifically blocks the binding of Sema4D to Plexin-B1. In vitro, we show that this antibody inhibits the suppressive effects of Sema4D on human osteoblast differentiation and mineralization. To test the therapeutic potential of the antibody in vivo, we generated a humanized mouse line, which expresses transgenic human Plexin-B1 instead of endogenous murine Plexin-B1. Employing these mice, we demonstrate that the anti-Plexin-B1 antibody exhibits beneficial effects in mouse models of postmenopausal osteoporosis and multiple sclerosis in vivo. In summary, our data identify an anti-Plexin-B1 antibody as a potential therapeutic agent for the treatment of osteoporosis and multiple sclerosis.


Antibodies, Monoclonal , Antigens, CD , Multiple Sclerosis , Nerve Tissue Proteins , Osteoporosis, Postmenopausal , Receptors, Cell Surface , Semaphorins , Animals , Antibodies, Monoclonal/therapeutic use , Antigens, CD/metabolism , Disease Models, Animal , Female , Humans , Ligands , Mice , Multiple Sclerosis/therapy , Nerve Tissue Proteins/antagonists & inhibitors , Nerve Tissue Proteins/metabolism , Osteoporosis, Postmenopausal/therapy , Receptors, Cell Surface/antagonists & inhibitors , Receptors, Cell Surface/metabolism , Semaphorins/antagonists & inhibitors , Semaphorins/metabolism
3.
mBio ; 13(1): e0350121, 2022 02 22.
Article En | MEDLINE | ID: mdl-35038903

Coenzyme F420 is a microbial redox cofactor that mediates diverse physiological functions and is increasingly used for biocatalytic applications. Recently, diversified biosynthetic routes to F420 and the discovery of a derivative, 3PG-F420, were reported. 3PG-F420 is formed via activation of 3-phospho-d-glycerate (3-PG) by CofC, but the structural basis of substrate binding, its evolution, as well as the role of CofD in substrate selection remained elusive. Here, we present a crystal structure of the 3-PG-activating CofC from Mycetohabitans sp. B3 and define amino acids governing substrate specificity. Site-directed mutagenesis enabled bidirectional switching of specificity and thereby revealed the short evolutionary trajectory to 3PG-F420 formation. Furthermore, CofC stabilized its product, thus confirming the structure of the unstable molecule and revealing its binding mode. The CofD enzyme was shown to significantly contribute to the selection of related intermediates to control the specificity of the combined biosynthetic CofC/D step. These results imply the need to change the design of combined CofC/D activity assays. Taken together, this work presents novel mechanistic and structural insights into 3PG-F420 biosynthesis and evolution and opens perspectives for the discovery and enhanced biotechnological production of coenzyme F420 derivatives in the future. IMPORTANCE The microbial cofactor F420 is crucial for processes like methanogenesis, antibiotics biosynthesis, drug resistance, and biocatalysis. Recently, a novel derivative of F420 (3PG-F420) was discovered, enabling the production and use of F420 in heterologous hosts. By analyzing the crystal structure of a CofC homolog whose substrate choice leads to formation of 3PG-F420, we defined amino acid residues governing the special substrate selectivity. A diagnostic residue enabled reprogramming of the substrate specificity, thus mimicking the evolution of the novel cofactor derivative. Furthermore, a labile reaction product of CofC was revealed that has not been directly detected so far. CofD was shown to provide another layer of specificity of the combined CofC/D reaction, thus controlling the initial substrate choice of CofC. The latter finding resolves a current debate in the literature about the starting point of F420 biosynthesis in various organisms.


Riboflavin , Riboflavin/metabolism , Oxidation-Reduction , Biocatalysis
4.
Biol Chem ; 403(2): 151-194, 2022 01 27.
Article En | MEDLINE | ID: mdl-34433238

The acetylation/acylation (ac(et)ylation) of lysine side chains is a dynamic post-translational modification (PTM) regulating fundamental cellular processes with implications on the organisms' ageing process: metabolism, transcription, translation, cell proliferation, regulation of the cytoskeleton and DNA damage repair. First identified to occur on histones, later studies revealed the presence of lysine ac(et)ylation in organisms of all kingdoms of life, in proteins covering all essential cellular processes. A remarkable finding showed that the NAD+-dependent sirtuin deacetylase Sir2 has an impact on replicative lifespan in Saccharomyces cerevisiae suggesting that lysine acetylation has a direct role in the ageing process. Later studies identified sirtuins as mediators for beneficial effects of caloric/dietary restriction on the organisms' health- or lifespan. However, the molecular mechanisms underlying these effects are only incompletely understood. Progress in mass-spectrometry, structural biology, synthetic and semi-synthetic biology deepened our understanding of this PTM. This review summarizes recent developments in the research field. It shows how lysine ac(et)ylation regulates protein function, how it is regulated enzymatically and non-enzymatically, how a dysfunction in this post-translational machinery contributes to disease development. A focus is set on sirtuins and lysine acyltransferases as these are direct sensors and mediators of the cellular metabolic state. Finally, this review highlights technological advances to study lysine ac(et)ylation.


Lysine , Sirtuins , Acetylation , Histones/metabolism , Lysine/metabolism , Saccharomyces cerevisiae/metabolism , Sirtuins/metabolism
5.
Curr Protoc ; 1(11): e277, 2021 Nov.
Article En | MEDLINE | ID: mdl-34748287

Proteins can be lysine-acetylated both enzymatically, by lysine acetyltransferases (KATs), and non-enzymatically, by acetyl-CoA and/or acetyl-phosphate. Such modification can be reversed by lysine deacetylases classified as NAD+ -dependent sirtuins or by classical Zn2+ -dependent deacetylases (KDACs). The regulation of protein lysine acetylation events by KATs and sirtuins/KDACs, or by non-enzymatic processes, is often assessed only indirectly by mass spectrometry or by mutational studies in cells. Mutational approaches to study lysine acetylation are limited, as these often poorly mimic lysine acetylation. Here, we describe protocols to assess the direct regulation of protein lysine acetylation by both sirtuins/KDACs and KATs, as well as non-enzymatically. We first describe a protocol for the production of site-specific lysine-acetylated proteins using a synthetic biological approach, the genetic code expansion concept (GCEC). These natively folded, lysine-acetylated proteins can then be used as direct substrates for sirtuins and KDACs. This approach addresses various limitations encountered with other methods. First, results from sirtuin/KDAC-catalyzed deacetylation assays obtained using acetylated peptides as substrates can vary considerably compared to experiments using natively folded substrate proteins. In addition, producing lysine-acetylated proteins for deacetylation assays by using recombinantly expressed KATs is difficult, as these often do not yield proteins that are homogeneously and quantitatively lysine acetylated. Moreover, KATs are often huge multi-domain proteins, which are difficult to recombinantly express and purify in soluble form. We also describe protocols to study the direct regulation of protein lysine acetylation, both enzymatically, by sirtuins/KDACs and KATs, and non-enzymatically, by acetyl-CoA and/or acetyl-phosphate. The latter protocol also includes a section that explains how specific lysine acetylation sites can be detected by liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS). The protocols described here can be useful for providing a more detailed understanding of the enzymatic and non-enzymatic regulation of lysine acetylation sites, an important aspect to judge their physiological significance. © 2021 The Authors. Current Protocols published by Wiley Periodicals LLC. Basic Protocol 1: Preparation of N-(ε)-lysine-acetylated proteins using the genetic code expansion concept (GCEC) Basic Protocol 2: In vitro sirtuin (SIRT)-catalyzed deacetylation of lysine-acetylated proteins prepared by the GCEC Basic Protocol 3: In vitro KDAC/HDAC-catalyzed deacetylation of lysine-acetylated proteins Basic Protocol 4: In vitro lysine acetylation of recombinantly expressed proteins by lysine acetyltransferases (KATs) Basic Protocol 5: In vitro non-enzymatic lysine acetylation of proteins by acetyl-CoA and/or acetyl-phosphate.


Lysine Acetyltransferases , Lysine , Acetylation , Chromatography, Liquid , Lysine/metabolism , Lysine Acetyltransferases/metabolism , Tandem Mass Spectrometry
6.
Proc Natl Acad Sci U S A ; 110(43): 17296-301, 2013 Oct 22.
Article En | MEDLINE | ID: mdl-24101465

Most secondary-active transporters transport their substrates using an electrochemical ion gradient. In contrast, the carnitine transporter (CaiT) is an ion-independent, l-carnitine/γ-butyrobetaine antiporter belonging to the betaine/carnitine/choline transporter family of secondary transporters. Recently determined crystal structures of CaiT from Escherichia coli and Proteus mirabilis revealed an inverted five-transmembrane-helix repeat similar to that in the amino acid/Na(+) symporter LeuT. The ion independence of CaiT makes it unique in this family. Here we show that mutations of arginine 262 (R262) make CaiT Na(+)-dependent. The transport activity of R262 mutants increased by 30-40% in the presence of a membrane potential, indicating substrate/Na(+) cotransport. Structural and biochemical characterization revealed that R262 plays a crucial role in substrate binding by stabilizing the partly unwound TM1' helix. Modeling CaiT from P. mirabilis in the outward-open and closed states on the corresponding structures of the related symporter BetP reveals alternating orientations of the buried R262 sidechain, which mimic sodium binding and unbinding in the Na(+)-coupled substrate symporters. We propose that a similar mechanism is operative in other Na(+)/H(+)-independent transporters, in which a positively charged amino acid replaces the cotransported cation. The oscillation of the R262 sidechain in CaiT indicates how a positive charge triggers the change between outward-open and inward-open conformations as a unifying critical step in LeuT-type transporters.


Antiporters/metabolism , Arginine/metabolism , Bacterial Proteins/metabolism , Proteus mirabilis/metabolism , Sodium/metabolism , Amino Acid Sequence , Antiporters/chemistry , Antiporters/genetics , Arginine/chemistry , Arginine/genetics , Bacterial Proteins/chemistry , Bacterial Proteins/genetics , Binding Sites/genetics , Biological Transport/genetics , Carnitine/chemistry , Carnitine/metabolism , Crystallography, X-Ray , Kinetics , Models, Molecular , Molecular Sequence Data , Mutation , Protein Binding , Protein Structure, Secondary , Protein Structure, Tertiary , Proteus mirabilis/chemistry , Proteus mirabilis/genetics , Sequence Homology, Amino Acid , Substrate Specificity
7.
Development ; 140(7): 1550-9, 2013 Apr.
Article En | MEDLINE | ID: mdl-23482490

Motile cilia perform crucial functions during embryonic development and throughout adult life. Development of organs containing motile cilia involves regulation of cilia formation (ciliogenesis) and formation of a luminal space (lumenogenesis) in which cilia generate fluid flows. Control of ciliogenesis and lumenogenesis is not yet fully understood, and it remains unclear whether these processes are coupled. In the zebrafish embryo, lethal giant larvae 2 (lgl2) is expressed prominently in ciliated organs. Lgl proteins are involved in establishing cell polarity and have been implicated in vesicle trafficking. Here, we identified a role for Lgl2 in development of ciliated epithelia in Kupffer's vesicle, which directs left-right asymmetry of the embryo; the otic vesicles, which give rise to the inner ear; and the pronephric ducts of the kidney. Using Kupffer's vesicle as a model ciliated organ, we found that depletion of Lgl2 disrupted lumen formation and reduced cilia number and length. Immunofluorescence and time-lapse imaging of Kupffer's vesicle morphogenesis in Lgl2-deficient embryos suggested cell adhesion defects and revealed loss of the adherens junction component E-cadherin at lateral membranes. Genetic interaction experiments indicate that Lgl2 interacts with Rab11a to regulate E-cadherin and mediate lumen formation that is uncoupled from cilia formation. These results uncover new roles and interactions for Lgl2 that are crucial for both lumenogenesis and ciliogenesis and indicate that these processes are genetically separable in zebrafish.


Cilia/physiology , Kupffer Cells/physiology , Morphogenesis/genetics , Zebrafish Proteins/physiology , Zebrafish , Animals , Animals, Genetically Modified , Body Patterning/genetics , Cell Polarity/genetics , Cilia/genetics , Cilia/metabolism , Embryo, Nonmammalian , Embryonic Development/genetics , Embryonic Development/physiology , Gene Expression Regulation, Developmental , Kupffer Cells/metabolism , Larva/genetics , Larva/metabolism , Morphogenesis/physiology , Zebrafish/embryology , Zebrafish/genetics , Zebrafish Proteins/genetics , Zebrafish Proteins/metabolism
9.
Nature ; 467(7312): 233-6, 2010 Sep 09.
Article En | MEDLINE | ID: mdl-20829798

Transport of solutes across biological membranes is performed by specialized secondary transport proteins in the lipid bilayer, and is essential for life. Here we report the structures of the sodium-independent carnitine/butyrobetaine antiporter CaiT from Proteus mirabilis (PmCaiT) at 2.3-A and from Escherichia coli (EcCaiT) at 3.5-A resolution. CaiT belongs to the family of betaine/carnitine/choline transporters (BCCT), which are mostly Na(+) or H(+) dependent, whereas EcCaiT is Na(+) and H(+) independent. The three-dimensional architecture of CaiT resembles that of the Na(+)-dependent transporters LeuT and BetP, but in CaiT a methionine sulphur takes the place of the Na(+) ion to coordinate the substrate in the central transport site, accounting for Na(+)-independent transport. Both CaiT structures show the fully open, inward-facing conformation, and thus complete the set of functional states that describe the alternating access mechanism. EcCaiT contains two bound butyrobetaine substrate molecules, one in the central transport site, the other in an extracellular binding pocket. In the structure of PmCaiT, a tryptophan side chain occupies the transport site, and access to the extracellular site is blocked. Binding of both substrates to CaiT reconstituted into proteoliposomes is cooperative, with Hill coefficients up to 1.7, indicating that the extracellular site is regulatory. We propose a mechanism whereby the occupied regulatory site increases the binding affinity of the transport site and initiates substrate translocation.


Antiporters/chemistry , Antiporters/metabolism , Bacterial Proteins/chemistry , Bacterial Proteins/metabolism , Escherichia coli Proteins/chemistry , Escherichia coli Proteins/metabolism , Escherichia coli/metabolism , Proteus mirabilis/metabolism , Betaine/analogs & derivatives , Betaine/metabolism , Biological Transport , Carnitine/metabolism , Models, Molecular
10.
PLoS Biol ; 7(4): e94, 2009 Apr 28.
Article En | MEDLINE | ID: mdl-19402751

PERIOD proteins are central components of the Drosophila and mammalian circadian clocks. The crystal structure of a Drosophila PERIOD (dPER) fragment comprising two PER-ARNT-SIM (PAS) domains (PAS-A and PAS-B) and two additional C-terminal alpha-helices (alphaE and alphaF) has revealed a homodimer mediated by intermolecular interactions of PAS-A with tryptophane 482 in PAS-B and helix alphaF. Here we present the crystal structure of a monomeric PAS domain fragment of dPER lacking the alphaF helix. Moreover, we have solved the crystal structure of a PAS domain fragment of the mouse PERIOD homologue mPER2. The mPER2 structure shows a different dimer interface than dPER, which is stabilized by interactions of the PAS-B beta-sheet surface including tryptophane 419 (equivalent to Trp482dPER). We have validated and quantitatively analysed the homodimer interactions of dPER and mPER2 by site-directed mutagenesis using analytical gel filtration, analytical ultracentrifugation, and co-immunoprecipitation experiments. Furthermore we show, by yeast-two-hybrid experiments, that the PAS-B beta-sheet surface of dPER mediates interactions with TIMELESS (dTIM). Our study reveals quantitative and qualitative differences between the homodimeric PAS domain interactions of dPER and its mammalian homologue mPER2. In addition, we identify the PAS-B beta-sheet surface as a versatile interaction site mediating mPER2 homodimerization in the mammalian system and dPER-dTIM heterodimer formation in the Drosophila system.


Cell Cycle Proteins/chemistry , Circadian Rhythm , Drosophila Proteins/chemistry , Drosophila/chemistry , Nuclear Proteins/chemistry , Protein Multimerization , Protein Structure, Tertiary , Transcription Factors/chemistry , Animals , Biological Clocks , Drosophila/metabolism , Drosophila Proteins/metabolism , Mice , Nuclear Proteins/metabolism , Period Circadian Proteins , Protein Structure, Secondary , Sequence Alignment , Tryptophan/metabolism
11.
Mol Cell ; 17(1): 69-82, 2005 Jan 07.
Article En | MEDLINE | ID: mdl-15629718

PERIOD proteins are central components of the Drosophila and mammalian circadian clock. Their function is controlled by daily changes in synthesis, cellular localization, phosphorylation, degradation, as well as specific interactions with other clock components. Here we present the crystal structure of a Drosophila PERIOD (dPER) fragment comprising two tandemly organized PAS (PER-ARNT-SIM) domains (PAS-A and PAS-B) and two additional C-terminal alpha helices (alphaE and alphaF). Our analysis reveals a noncrystallographic dPER dimer mediated by intermolecular interactions of PAS-A with PAS-B and helix alphaF. We show that alphaF is essential for dPER homodimerization and that the PAS-A-alphaF interaction plays a crucial role in dPER clock function, as it is affected by the 29 hr long-period perL mutation.


Drosophila Proteins/chemistry , Nuclear Proteins/chemistry , Amino Acid Sequence , Animals , Circadian Rhythm , Crystallography, X-Ray , Dimerization , Drosophila/chemistry , Drosophila/genetics , Drosophila/physiology , Drosophila Proteins/genetics , Drosophila Proteins/physiology , Models, Molecular , Molecular Sequence Data , Mutation , Nuclear Proteins/genetics , Nuclear Proteins/physiology , Period Circadian Proteins , Protein Structure, Quaternary , Protein Structure, Tertiary , Repetitive Sequences, Amino Acid , Sequence Homology, Amino Acid , Static Electricity
...