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1.
Microbiol Spectr ; 11(6): e0300323, 2023 Dec 12.
Artigo em Inglês | MEDLINE | ID: mdl-37796003

RESUMO

IMPORTANCE: This work has global significance in the catfish industry, which provides food for increasing global populations. E. ictaluri is a leading cause of disease loss, and EseN is an important player in E. ictaluri virulence. The E. ictaluri T3SS effector EseN plays an essential role in establishing infection, but the specific role EseN plays is not well characterized. EseN belongs to a family of phosphothreonine lyase effectors that specifically target host mitogen activated protein kinase (MAPK) pathways important in regulating host responses to infection. No phosphothreonine lyase equivalents are known in eukaryotes, making this family of effectors an attractive target for indirect narrow-spectrum antibiotics. Targeting of major vault protein and PDK1 kinase by EseN has not been reported in EseN homologs in other pathogens and may indicate unique functions of E. ictaluri EseN. EseN targeting of PDK1 is particularly interesting in that it is linked to an extraordinarily diverse group of cellular functions.


Assuntos
Infecções por Enterobacteriaceae , Doenças dos Peixes , Liases , Animais , Edwardsiella ictaluri/fisiologia , Fosfotreonina , Sistema de Sinalização das MAP Quinases , Macrófagos , Morte Celular
2.
ACS Chem Biol ; 18(9): 1938-1958, 2023 09 15.
Artigo em Inglês | MEDLINE | ID: mdl-37595155

RESUMO

Phosphorylation and dephosphorylation of proteins by kinases and phosphatases are central to cellular responses and function. The structural effects of serine and threonine phosphorylation were examined in peptides and in proteins, by circular dichroism, NMR spectroscopy, bioinformatics analysis of the PDB, small-molecule X-ray crystallography, and computational investigations. Phosphorylation of both serine and threonine residues induces substantial conformational restriction in their physiologically more important dianionic forms. Threonine exhibits a particularly strong disorder-to-order transition upon phosphorylation, with dianionic phosphothreonine preferentially adopting a cyclic conformation with restricted ϕ (ϕ ∼ -60°) stabilized by three noncovalent interactions: a strong intraresidue phosphate-amide hydrogen bond, an n → π* interaction between consecutive carbonyls, and an n → σ* interaction between the phosphate Oγ lone pair and the antibonding orbital of C-Hß that restricts the χ2 side-chain conformation. Proline is unique among the canonical amino acids for its covalent cyclization on the backbone. Phosphothreonine can mimic proline's backbone cyclization via noncovalent interactions. The preferred torsions of dianionic phosphothreonine are ϕ,ψ = polyproline II helix > α-helix (ϕ ∼ -60°); χ1 = g-; χ2 ∼ +115° (eclipsed C-H/O-P bonds). This structural signature is observed in diverse proteins, including in the activation loops of protein kinases and in protein-protein interactions. In total, these results suggest a structural basis for the differential use and evolution of threonine versus serine phosphorylation sites in proteins, with serine phosphorylation typically inducing smaller, rheostat-like changes, versus threonine phosphorylation promoting larger, step function-like switches, in proteins.


Assuntos
Serina , Treonina , Fosfotreonina , Fosforilação , Aminoácidos
3.
Int J Mol Sci ; 24(5)2023 Mar 01.
Artigo em Inglês | MEDLINE | ID: mdl-36902219

RESUMO

Identification of specific protein phosphatase-1 (PP1) inhibitors is of special importance regarding the study of its cellular functions and may have therapeutic values in diseases coupled to signaling processes. In this study, we prove that a phosphorylated peptide of the inhibitory region of myosin phosphatase (MP) target subunit (MYPT1), R690QSRRS(pT696)QGVTL701 (P-Thr696-MYPT1690-701), interacts with and inhibits the PP1 catalytic subunit (PP1c, IC50 = 3.84 µM) and the MP holoenzyme (Flag-MYPT1-PP1c, IC50 = 3.84 µM). Saturation transfer difference NMR measurements established binding of hydrophobic and basic regions of P-Thr696-MYPT1690-701 to PP1c, suggesting interactions with the hydrophobic and acidic substrate binding grooves. P-Thr696-MYPT1690-701 was dephosphorylated by PP1c slowly (t1/2 = 81.6-87.9 min), which was further impeded (t1/2 = 103 min) in the presence of the phosphorylated 20 kDa myosin light chain (P-MLC20). In contrast, P-Thr696-MYPT1690-701 (10-500 µM) slowed down the dephosphorylation of P-MLC20 (t1/2 = 1.69 min) significantly (t1/2 = 2.49-10.06 min). These data are compatible with an unfair competition mechanism between the inhibitory phosphopeptide and the phosphosubstrate. Docking simulations of the PP1c-P-MYPT1690-701 complexes with phosphothreonine (PP1c-P-Thr696-MYPT1690-701) or phosphoserine (PP1c-P-Ser696-MYPT1690-701) suggested their distinct poses on the surface of PP1c. In addition, the arrangements and distances of the surrounding coordinating residues of PP1c around the phosphothreonine or phosphoserine at the active site were distinct, which may account for their different hydrolysis rate. It is presumed that P-Thr696-MYPT1690-701 binds tightly at the active center but the phosphoester hydrolysis is less preferable compared to P-Ser696-MYPT1690-701 or phosphoserine substrates. Moreover, the inhibitory phosphopeptide may serve as a template to synthesize cell permeable PP1-specific peptide inhibitors.


Assuntos
Inibidores Enzimáticos , Fosfopeptídeos , Proteína Fosfatase 1 , Fosfatase de Miosina-de-Cadeia-Leve/metabolismo , Fosfopeptídeos/química , Fosfopeptídeos/farmacologia , Fosforilação , Fosfosserina/metabolismo , Fosfotreonina/metabolismo , Proteína Fosfatase 1/antagonistas & inibidores , Proteína Fosfatase 1/metabolismo , Inibidores Enzimáticos/química , Inibidores Enzimáticos/farmacologia
4.
Nature ; 613(7945): 759-766, 2023 01.
Artigo em Inglês | MEDLINE | ID: mdl-36631611

RESUMO

Protein phosphorylation is one of the most widespread post-translational modifications in biology1,2. With advances in mass-spectrometry-based phosphoproteomics, 90,000 sites of serine and threonine phosphorylation have so far been identified, and several thousand have been associated with human diseases and biological processes3,4. For the vast majority of phosphorylation events, it is not yet known which of the more than 300 protein serine/threonine (Ser/Thr) kinases encoded in the human genome are responsible3. Here we used synthetic peptide libraries to profile the substrate sequence specificity of 303 Ser/Thr kinases, comprising more than 84% of those predicted to be active in humans. Viewed in its entirety, the substrate specificity of the kinome was substantially more diverse than expected and was driven extensively by negative selectivity. We used our kinome-wide dataset to computationally annotate and identify the kinases capable of phosphorylating every reported phosphorylation site in the human Ser/Thr phosphoproteome. For the small minority of phosphosites for which the putative protein kinases involved have been previously reported, our predictions were in excellent agreement. When this approach was applied to examine the signalling response of tissues and cell lines to hormones, growth factors, targeted inhibitors and environmental or genetic perturbations, it revealed unexpected insights into pathway complexity and compensation. Overall, these studies reveal the intrinsic substrate specificity of the human Ser/Thr kinome, illuminate cellular signalling responses and provide a resource to link phosphorylation events to biological pathways.


Assuntos
Fosfoproteínas , Proteínas Serina-Treonina Quinases , Proteoma , Serina , Treonina , Humanos , Fosforilação , Proteínas Serina-Treonina Quinases/metabolismo , Serina/metabolismo , Especificidade por Substrato , Treonina/metabolismo , Proteoma/química , Proteoma/metabolismo , Conjuntos de Dados como Assunto , Fosfoproteínas/química , Fosfoproteínas/metabolismo , Linhagem Celular , Fosfosserina/metabolismo , Fosfotreonina/metabolismo
5.
Nat Commun ; 13(1): 7226, 2022 11 24.
Artigo em Inglês | MEDLINE | ID: mdl-36433969

RESUMO

Protein phosphorylation is a ubiquitous post-translational modification used to regulate cellular processes and proteome architecture by modulating protein-protein interactions. The identification of phosphorylation events through proteomic surveillance has dramatically outpaced our capacity for functional assignment using traditional strategies, which often require knowledge of the upstream kinase a priori. The development of phospho-amino-acid-specific orthogonal translation systems, evolutionarily divergent aminoacyl-tRNA synthetase and tRNA pairs that enable co-translational insertion of a phospho-amino acids, has rapidly improved our ability to assess the physiological function of phosphorylation by providing kinase-independent methods of phosphoprotein production. Despite this utility, broad deployment has been hindered by technical limitations and an inability to reconstruct complex phopho-regulatory networks. Here, we address these challenges by optimizing genetically encoded phosphothreonine translation to characterize phospho-dependent kinase activation mechanisms and, subsequently, develop a multi-level protein interaction platform to directly assess the overlap of kinase and phospho-binding protein substrate networks with phosphosite-level resolution.


Assuntos
Aminoacil-tRNA Sintetases , Proteoma , Humanos , Fosfotreonina , Proteoma/genética , Proteômica , Aminoacil-tRNA Sintetases/genética , Aminoacil-tRNA Sintetases/metabolismo , RNA de Transferência/metabolismo
6.
PLoS One ; 17(10): e0275860, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-36227898

RESUMO

Dual specificity phosphatase 7 (DUSP7) is a protein belonging to a broad group of phosphatases that can dephosphorylate phosphoserine/phosphothreonine as well as phosphotyrosine residues within the same substrate. DUSP7 has been linked to the negative regulation of mitogen activated protein kinases (MAPK), and in particular to the regulation of extracellular signal-regulated kinases 1 and 2 (ERK1/2). MAPKs play an important role in embryonic development, where their duration, magnitude, and spatiotemporal activity must be strictly controlled by other proteins, among others by DUSPs. In this study, we focused on the effect of DUSP7 depletion on the in vitro differentiation of mouse embryonic stem (ES) cells. We showed that even though DUSP7 knock-out ES cells do retain some of their basic characteristics, when it comes to differentiation, they preferentially differentiate towards neural cells, while the formation of early cardiac mesoderm is repressed. Therefore, our data indicate that DUSP7 is necessary for the correct formation of neuroectoderm and cardiac mesoderm during the in vitro differentiation of ES cells.


Assuntos
Fosfatases de Especificidade Dupla/metabolismo , Células-Tronco Embrionárias Murinas , Animais , Fosfatase 1 de Especificidade Dupla/metabolismo , Fosfatases de Especificidade Dupla/genética , Mesoderma/metabolismo , Camundongos , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Células-Tronco Embrionárias Murinas/metabolismo , Fosfosserina , Fosfotreonina , Fosfotirosina
7.
PLoS One ; 17(9): e0273797, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-36048825

RESUMO

There is growing evidence to suggest that phosphohistidines are present at significant levels in mammalian cells and play a part in regulating cellular activity, in particular signaling pathways related to cancer. Because of the chemical instability of phosphohistidine at neutral or acid pH, it remains unclear how much phosphohistidine is present in cells. Here we describe a protocol for extracting proteins from mammalian cells in a way that avoids loss of covalent phosphates from proteins, and use it to measure phosphohistidine concentrations in human bronchial epithelial cell (16HBE14o-) lysate using 31P NMR spectroscopic analysis. Phosphohistidine is determined on average to be approximately one third as abundant as phosphoserine and phosphothreonine combined (and thus roughly 15 times more abundant than phosphotyrosine). The amount of phosphohistidine, and phosphoserine/phosphothreonine per gram of protein from a cell lysate was determined to be 23 µmol/g and 68 µmol/g respectively. The amount of phosphohistidine, and phosphoserine/phosphothreonine per cell was determined to be 1.8 fmol/cell, and 5.8 fmol/cell respectively. Phosphorylation is largely at the N3 (tele) position. Typical tryptic digest conditions result in loss of most of the phosphohistidine present, which may explain why the amounts reported here are greater than is generally seen using mass spectroscopy assays. The results further strengthen the case for a functional role of phosphohistidine in eukaryotic cells.


Assuntos
Histidina , Proteínas , Animais , Linhagem Celular , Histidina/análogos & derivados , Histidina/metabolismo , Humanos , Mamíferos/metabolismo , Fosforilação , Fosfosserina/metabolismo , Fosfotreonina/metabolismo , Proteínas/metabolismo
8.
Angew Chem Int Ed Engl ; 61(35): e202208295, 2022 08 26.
Artigo em Inglês | MEDLINE | ID: mdl-35793207

RESUMO

Catalysis by radical enzymes dependent on coenzyme B12 (AdoCbl) relies on the reactive primary 5'-deoxy-5'adenosyl radical, which originates from reversible Co-C bond homolysis of AdoCbl. This bond homolysis is accelerated roughly 1012 -fold upon binding the enzyme substrate. The structural basis for this activation is still strikingly enigmatic. As revealed here, a displaced firm adenosine binding cavity in substrate-loaded glutamate mutase (GM) causes a structural misfit for intact AdoCbl that is relieved by the homolytic Co-C bond cleavage. Strategically interacting adjacent adenosine- and substrate-binding protein cavities provide a tight caged radical reaction space, controlling the entire radical path. The GM active site is perfectly structured for promoting radical catalysis, including "negative catalysis", a paradigm for AdoCbl-dependent mutases.


Assuntos
Cobamidas , Transferases Intramoleculares , Adenosina , Catálise , Cobamidas/química , Transferases Intramoleculares/metabolismo , Metilmalonil-CoA Mutase/química , Metilmalonil-CoA Mutase/metabolismo , Fosfotreonina/análogos & derivados
9.
J Photochem Photobiol B ; 232: 112471, 2022 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-35644067

RESUMO

The CarH photoreceptor exploits of the light-sensing ability of coenzyme B12 ( adenosylcobalamin = AdoCbl) to perform its catalytic function, which includes large-scale structural changes to regulate transcription. In daylight, transcription is activated in CarH via the photo-cleavage of the Co-C5' bond of coenzyme B12. Subsequently, the photoproduct, 4',5'-anhydroadenosine (anhAdo) is formed inducing dissociation of the CarH tetramer from DNA. Several experimental studies have proposed that hydridocoblamin (HCbl) may be formed in process with anhAdo. The photolytic cleavage of the Co-C5' bond of AdoCbl was previously investigated using photochemical techniques and the involvement of both singlet and triplet excited states were explored. Herein, QM/MM calculations were employed to probe (1) the photolytic processes which may involve singlet excited states, (2) the mechanism of anhAdo formation, and (3) whether HCbl is a viable intermediate in CarH. Time-dependent density functional theory (TD-DFT) calculations indicate that the mechanism of photodissociation of the Ado ligand involves the ligand field (LF) portion of the lowest singlet excited state (S1) potential energy surface (PES). This is followed by deactivation to a point on the S0 PES where the Co-C5' bond remains broken. This species corresponds to a singlet diradical intermediate. From this point, the PES for anhAdo formation was explored, using the Co-C5' and Co-C4' bond distances as active coordinates, and a local minimum representing anhAdo and HCbl formation was found. The transition state (TS) for the formation of the Co-H bond of HCbl was located and its identity was confirmed by a single imaginary frequency of i1592 cm-1. Comparisons to experimental studies and the potential role of rotation around the N-glycosidic bond of the Ado ligand were discussed.


Assuntos
Cobamidas , Cobamidas/química , Ligantes , Fosfotreonina/análogos & derivados
10.
Methods Enzymol ; 669: 229-259, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-35644173

RESUMO

Coenzyme B12 (adenosylcobalamin) -dependent ethanolamine ammonia-lyase (EAL) is the signature enzyme in ethanolamine utilization metabolism associated with microbiome homeostasis and disease conditions in the human gut. The enzyme conducts a complex choreography of bond-making/bond-breaking steps that rearrange substrate to products through a radical mechanism, with themes common to other coenzyme B12-dependent and radical enzymes. The methods presented are targeted to test the hypothesis that particular, select protein and coupled solvent configurational fluctuations contribute to enzyme function. The general approach is to correlate enzyme function with an introduced perturbation that alters the properties (for example, degree of concertedness, or collectiveness) of protein and coupled solvent dynamics. Methods for sample preparation and low-temperature kinetic measurements by using temperature-step reaction initiation and time-resolved, full-spectrum electron paramagnetic resonance spectroscopy are detailed. A framework for interpretation of results obtained in ensemble systems under conditions of statistical equilibrium within the reacting, globally unstable state is presented. The temperature-dependence of the first-order rate constants for decay of the cryotrapped paramagnetic substrate radical state in EAL, through the chemical step of radical rearrangement, displays a piecewise-continuous Arrhenius dependence from 203 to 295K, punctuated by a kinetic bifurcation over 219-220K. The results reveal the obligatory contribution of a class of select collective protein and coupled solvent fluctuations to the interconversion of two resolved, sequential configurational substates, on the decay time scale. The select class of collective fluctuations also contributes to the chemical step. The methods and analysis are generally applicable to other coenzyme B12-dependent and related radical enzymes.


Assuntos
Etanolamina Amônia-Liase , Catálise , Cobamidas , Etanolamina Amônia-Liase/química , Etanolamina Amônia-Liase/metabolismo , Humanos , Fosfotreonina/análogos & derivados , Salmonella typhimurium/metabolismo , Solventes/química
11.
Methods Enzymol ; 668: 243-284, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-35589195

RESUMO

Adenosylcobalamin (AdoCbl) or coenzyme B12-dependent enzymes tend to undergo mechanism-based inactivation during catalysis or inactivation in the absence of substrate. Such inactivation may be inevitable because they use a highly reactive radical for catalysis, and side reactions of radical intermediates result in the damage of the coenzyme. How do living organisms address such inactivation when enzymes are inactivated by undesirable side reactions? We discovered reactivating factors for radical B12 eliminases. They function as releasing factors for damaged cofactor(s) from enzymes and thus mediate their exchange for intact AdoCbl. Since multiple turnovers and chaperone functions were demonstrated, they were renamed "reactivases" or "reactivating chaperones." They play an essential role in coenzyme recycling as part of the activity-maintaining systems for B12 enzymes. In this chapter, we describe our investigations on reactivating chaperones, including their discovery, gene cloning, preparation, characterization, activity assays, and mechanistic studies, that have been conducted using a wide range of biochemical and structural methods that we have developed.


Assuntos
Etanolamina Amônia-Liase , Propanodiol Desidratase , Cobamidas/química , Coenzimas , Etanolamina Amônia-Liase/química , Glicerol , Hidroliases , Chaperonas Moleculares , Fosfotreonina/análogos & derivados , Propanodiol Desidratase/química , Propanodiol Desidratase/genética
12.
Methods Enzymol ; 668: 181-242, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-35589194

RESUMO

Adenosylcobalamin (AdoCbl) or coenzyme B12-dependent enzymes catalyze intramolecular group-transfer reactions and ribonucleotide reduction in a wide variety of organisms from bacteria to animals. They use a super-reactive primary-carbon radical formed by the homolysis of the coenzyme's Co-C bond for catalysis and thus belong to the larger class of "radical enzymes." For understanding the general mechanisms of radical enzymes, it is of great importance to establish the general mechanism of AdoCbl-dependent catalysis using enzymes that catalyze the simplest reactions-such as diol dehydratase, glycerol dehydratase and ethanolamine ammonia-lyase. These enzymes are often called "eliminases." We have studied AdoCbl and eliminases for more than a half century. Progress has always been driven by the development of new experimental methodologies. In this chapter, we describe our investigations on these enzymes, including their metabolic roles, gene cloning, preparation, characterization, activity assays, and mechanistic studies, that have been conducted using a wide range of biochemical and structural methodologies we have developed.


Assuntos
Etanolamina Amônia-Liase , Animais , Cobamidas/química , Cobamidas/metabolismo , Etanolamina Amônia-Liase/química , Etanolamina Amônia-Liase/metabolismo , Glicerol , Hidroliases , Fosfotreonina/análogos & derivados
13.
Methods Enzymol ; 668: 349-372, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-35589201

RESUMO

Coenzyme B12 is one of the most complex cofactors found in nature and synthesized de novo by certain groups of bacteria. Although its use in various enzymatic reactions is well characterized, only recently an unusual light-sensing function has been ascribed to coenzyme B12. It has been reported that the coenzyme B12 binding protein CarH, found in the carotenoid biosynthesis pathway of several thermostable bacteria, binds to the promoter region of DNA and suppresses transcription. To overcome the harmful effects of light-induced damage in the cells, CarH releases DNA in the presence of light and promotes transcription and synthesis of carotenoids, thereby working as a photoreceptor. CarH is able to achieve this by exploiting the photosensitive nature of the CoC bond between the adenosyl moiety and the cobalt atom in the coenzyme B12 molecule. Extensive structural and spectroscopy studies provided a mechanistic understanding of the molecular basis of this unique light-sensitive reaction. Most studies on CarH have used the ortholog from the thermostable bacterium Thermus thermophilus, due to the ease with which it can be expressed and purified in high quantities. In this chapter we give an overview of this intriguing class of photoreceptors and report a step-by-step protocol for expression, purification and spectroscopy experiments (both static and time-resolved techniques) employed in our laboratory to study CarH from T. thermophilus. We hope the contents of this chapter will be of interest to the wider coenzyme B12 community and apprise them of the potential and possibilities of using coenzyme B12 as a light-sensing probe in a protein scaffold.


Assuntos
Proteínas de Bactérias , Regulação Bacteriana da Expressão Gênica , Proteínas de Bactérias/metabolismo , Cobamidas/química , Cobamidas/genética , Cobamidas/metabolismo , DNA/metabolismo , Fosfotreonina/análogos & derivados , Thermus thermophilus/genética , Thermus thermophilus/metabolismo , Vitamina B 12/metabolismo
14.
ACS Chem Biol ; 17(2): 414-425, 2022 02 18.
Artigo em Inglês | MEDLINE | ID: mdl-35129954

RESUMO

Site-specific modification of proteins has important applications in biological research and drug development. Reactive tags such as azide, alkyne, and tetrazine have been used extensively to achieve the abovementioned goal. However, bulky side-chain "ligation scars" are often left after the labeling and may hinder the biological application of such engineered protein products. Conjugation chemistry via dehydroalanine (Dha) may provide an opportunity for "traceless" ligation because the activated alkene moiety on Dha can then serve as an electrophile to react with radicalophile, thiol/amine nucleophile, and reactive phosphine probe to introduce a minimal linker in the protein post-translational modifications. In this report, we present a mild and highly efficient enzymatic approach to incorporate Dha with phosphothreonine/serine lyases, OspF and SpvC. These lyases originally catalyze an irreversible elimination reaction that converts a doubly phosphorylated substrate with phosphothreonine (pT) or phosphoserine (pS) to dehydrobutyrine (Dhb) or Dha. To generate a simple monophosphorylated tag for these lyases, we conducted a systematic approach to profile the substrate specificity of OspF and SpvC using peptide arrays and self-assembled monolayers for matrix-assisted laser desorption/ionization mass spectrometry. The optimized tag, [F/Y/W]-pT/pS-[F/Y/W] (where [F/Y/W] indicates an aromatic residue), results in a ∼10-fold enhancement of the overall peptide labeling efficiency via Dha chemistry and enables the first demonstration of protein labeling as well as live cell labeling with a minimal ligation linker via enzyme-mediated incorporation of Dha.


Assuntos
Liases , Alanina/análogos & derivados , Alanina/química , Liases/metabolismo , Fosfotreonina/metabolismo , Processamento de Proteína Pós-Traducional , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz
15.
Environ Microbiol ; 24(4): 1865-1886, 2022 04.
Artigo em Inglês | MEDLINE | ID: mdl-35005822

RESUMO

Light-induced carotenogenesis in Myxococcus xanthus is controlled by the B12 -based CarH repressor and photoreceptor, and by a separate intricate pathway involving singlet oxygen, the B12 -independent CarH paralogue CarA and various other proteins, some eukaryotic-like. Whether other myxobacteria conserve these pathways and undergo photoregulated carotenogenesis is unknown. Here, comparative analyses across 27 Myxococcales genomes identified carotenogenic genes, albeit arranged differently, with carH often in their genomic vicinity, in all three Myxococcales suborders. However, CarA and its associated factors were found exclusively in suborder Cystobacterineae, with carA-carH invariably in tandem in a syntenic carotenogenic operon, except for Cystobacter/Melittangium, which lack CarA but retain all other factors. We experimentally show B12 -mediated photoregulated carotenogenesis in representative myxobacteria, and a remarkably plastic CarH operator design and DNA binding across Myxococcales. Unlike the two characterized CarH from other phyla, which are tetrameric, Cystobacter CarH (the first myxobacterial homologue amenable to analysis in vitro) is a dimer that combines direct CarH-like B12 -based photoregulation with CarA-like DNA binding and inhibition by an antirepressor. This study provides new molecular insights into B12 -dependent photoreceptors. It further establishes the B12 -dependent pathway for photoregulated carotenogenesis as broadly prevalent across myxobacteria and its evolution, exclusively in one suborder, into a parallel complex B12 -independent circuit.


Assuntos
Regulação Bacteriana da Expressão Gênica , Myxococcales , Proteínas de Bactérias/metabolismo , DNA/metabolismo , Myxococcales/genética , Myxococcales/metabolismo , Fosfotreonina/análogos & derivados , Proteínas Repressoras/metabolismo
16.
Proc Natl Acad Sci U S A ; 118(24)2021 06 15.
Artigo em Inglês | MEDLINE | ID: mdl-34099554

RESUMO

Differential concentrations of phytohormone trigger distinct outputs, which provides a mechanism for the plasticity of plant development and an adaptation strategy among plants to changing environments. However, the underlying mechanisms of the differential responses remain unclear. Here we report that a high concentration of auxin, distinct from the effect of low auxin concentration, enhances abscisic acid (ABA) responses in Arabidopsis thaliana, which partially relies on TRANS-MEMBERANE KINASE 1 (TMK1), a key regulator in auxin signaling. We show that high auxin and TMK1 play essential and positive roles in ABA signaling through regulating ABA INSENSITIVE 1 and 2 (ABI1/2), two negative regulators of the ABA pathway. TMK1 inhibits the phosphatase activity of ABI2 by direct phosphorylation of threonine 321 (T321), a conserved phosphorylation site in ABI2 proteins, whose phosphorylation status is important for both auxin and ABA responses. This TMK1-dependent auxin signaling in the regulation of ABA responses provides a possible mechanism underlying the high auxin responses in plants and an alternative mechanism involved in the coordination between auxin and ABA signaling.


Assuntos
Ácido Abscísico/metabolismo , Proteínas de Arabidopsis/metabolismo , Arabidopsis/metabolismo , Ácidos Indolacéticos/metabolismo , Fosfoproteínas Fosfatases/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Transdução de Sinais , Epistasia Genética , Fosforilação , Fosfotreonina/metabolismo , Ligação Proteica
17.
Chemistry ; 27(29): 7930-7941, 2021 May 20.
Artigo em Inglês | MEDLINE | ID: mdl-33792120

RESUMO

Diol dehydratase, dependent on coenzyme B12 (B12 -dDDH), displays a peculiar feature of being inactivated by its native substrate glycerol (GOL). Surprisingly, the isofunctional enzyme, B12 -independent glycerol dehydratase (B12 -iGDH), does not undergo suicide inactivation by GOL. Herein we present a series of QM/MM and MD calculations aimed at understanding the mechanisms of substrate-induced suicide inactivation in B12 -dDDH and that of resistance of B12 -iGDH to inactivation. We show that the first step in the enzymatic transformation of GOL, hydrogen abstraction, can occur from both ends of the substrate (either C1 or C3 of GOL). Whereas C1 abstraction in both enzymes leads to product formation, C3 abstraction in B12 -dDDH results in the formation of a low energy radical intermediate, which is effectively trapped within a deep well on the potential energy surface. The long lifetime of this radical intermediate likely enables its side reactions, leading to inactivation. In B12 -iGDH, by comparison, C3 abstraction is an endothermic step; consequently, the resultant radical intermediate is not of low energy, and the reverse process of reforming the reactant is possible.


Assuntos
Propanodiol Desidratase , Cobamidas , Glicerol , Humanos , Hidroliases , Fosfotreonina/análogos & derivados
18.
Mol Brain ; 14(1): 66, 2021 04 08.
Artigo em Inglês | MEDLINE | ID: mdl-33832520

RESUMO

GAP-43 is a vertebrate neuron-specific protein and that is strongly related to axon growth and regeneration; thus, this protein has been utilized as a classical molecular marker of these events and growth cones. Although GAP-43 was biochemically characterized more than a quarter century ago, how this protein is related to these events is still not clear. Recently, we identified many phosphorylation sites in the growth cone membrane proteins of rodent brains. Two phosphorylation sites of GAP-43, S96 and T172, were found within the top 10 hit sites among all proteins. S96 has already been characterized (Kawasaki et al., 2018), and here, phosphorylation of T172 was characterized. In vitro (cultured neurons) and in vivo, an antibody specific to phosphorylated T172 (pT172 antibody) specifically recognized cultured growth cones and growing axons in developing mouse neurons, respectively. Immunoblotting showed that pT172 antigens were more rapidly downregulated throughout development than those of pS96 antibody. From the primary structure, this phosphorylation site was predicted to be conserved in a wide range of animals including primates. In the developing marmoset brainstem and in differentiated neurons derived from human induced pluripotent stem cells, immunoreactivity with pT172 antibody revealed patterns similar to those in mice. pT172 antibody also labeled regenerating axons following sciatic nerve injury. Taken together, the T172 residue is widely conserved in a wide range of mammals including primates, and pT172 is a new candidate molecular marker for growing axons.


Assuntos
Axônios/metabolismo , Biomarcadores/metabolismo , Proteína GAP-43/metabolismo , Mamíferos/metabolismo , Fosfotreonina/metabolismo , Sequência de Aminoácidos , Animais , Anticorpos/metabolismo , Encéfalo/embriologia , Callithrix , Células Cultivadas , Furões , Proteína GAP-43/química , Cones de Crescimento/metabolismo , Células HEK293 , Humanos , Proteínas Quinases JNK Ativadas por Mitógeno/metabolismo , Camundongos Endogâmicos C57BL , Regeneração Nervosa , Fosforilação , Primatas , Nervo Isquiático/lesões
19.
Plant Cell ; 33(5): 1790-1812, 2021 07 02.
Artigo em Inglês | MEDLINE | ID: mdl-33630095

RESUMO

Calcium (Ca2+)/calmodulin (CaM)-dependent protein kinase (CCaMK) is an important positive regulator of abscisic acid (ABA) and abiotic stress signaling in plants and is believed to act upstream of mitogen-activated protein kinase (MAPK) in ABA signaling. However, it is unclear how CCaMK activates MAPK in ABA signaling. Here, we show that OsDMI3, a rice (Oryza sativa) CCaMK, directly interacts with and phosphorylates OsMKK1, a MAPK kinase (MKK) in rice, in vitro and in vivo. OsDMI3 was found to directly phosphorylate Thr-25 in the N-terminus of OsMKK1, and this Thr-25 phosphorylation is OsDMI3-specific in ABA signaling. The activation of OsMKK1 and its downstream kinase OsMPK1 is dependent on Thr-25 phosphorylation of OsMKK1 in ABA signaling. Moreover, ABA treatment induces phosphorylation in the activation loop of OsMKK1, and the two phosphorylations, in the N-terminus and in the activation loop, are independent. Further analyses revealed that OsDMI3-mediated phosphorylation of OsMKK1 positively regulates ABA responses in seed germination, root growth, and tolerance to both water stress and oxidative stress. Our results indicate that OsMKK1 is a direct target of OsDMI3, and OsDMI3-mediated phosphorylation of OsMKK1 plays an important role in activating the MAPK cascade and ABA signaling.


Assuntos
Ácido Abscísico/metabolismo , Proteínas Quinases Dependentes de Cálcio-Calmodulina/metabolismo , Cálcio/metabolismo , Quinases de Proteína Quinase Ativadas por Mitógeno/metabolismo , Oryza/enzimologia , Proteínas de Plantas/metabolismo , Ácido Abscísico/farmacologia , Quinases de Proteína Quinase Ativadas por Mitógeno/química , Modelos Biológicos , Oryza/efeitos dos fármacos , Oryza/fisiologia , Estresse Oxidativo/efeitos dos fármacos , Fosforilação/efeitos dos fármacos , Fosfotreonina/metabolismo , Ligação Proteica/efeitos dos fármacos , Transdução de Sinais/efeitos dos fármacos , Estresse Fisiológico/efeitos dos fármacos , Água
20.
Cells ; 10(2)2021 02 09.
Artigo em Inglês | MEDLINE | ID: mdl-33572403

RESUMO

Septins are GTP-binding proteins that form heteromeric filaments for proper cell growth and migration. Among the septins, septin7 (SEPT7) is an important component of all septin filaments. Here we show that protein kinase A (PKA) phosphorylates SEPT7 at Thr197, thus disrupting septin filament dynamics and ciliogenesis. The Thr197 residue of SEPT7, a PKA phosphorylating site, was conserved among different species. Treatment with cAMP or overexpression of PKA catalytic subunit (PKACA2) induced SEPT7 phosphorylation, followed by disruption of septin filament formation. Constitutive phosphorylation of SEPT7 at Thr197 reduced SEPT7‒SEPT7 interaction, but did not affect SEPT7‒SEPT6‒SEPT2 or SEPT4 interaction. Moreover, we noted that SEPT7 interacted with PKACA2 via its GTP-binding domain. Furthermore, PKA-mediated SEPT7 phosphorylation disrupted primary cilia formation. Thus, our data uncover the novel biological function of SEPT7 phosphorylation in septin filament polymerization and primary cilia formation.


Assuntos
Proteínas de Ciclo Celular/metabolismo , Cílios/metabolismo , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Organogênese , Septinas/metabolismo , Sequência de Aminoácidos , Proteínas de Ciclo Celular/química , Sequência Conservada , Humanos , Fosforilação , Fosfotreonina/metabolismo , Ligação Proteica , Domínios Proteicos , Septinas/química , Especificidade da Espécie
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