Adenosine undergoes ATP-dependent phosphorylation catalyzed by adenosine kinase (ADK). In plants, ADK also phosphorylates cytokinin ribosides, transport forms of the hormone. Here, we investigated the substrate preferences, oligomeric states, and structures of ADKs from moss (Physcomitrella patens) and maize (Zea mays) alongside metabolomic and phenotypic analyses. We showed that dexamethasone-inducible ZmADK overexpressor lines in Arabidopsis can benefit from a higher number of lateral roots and larger root areas under nitrogen starvation. We discovered that maize and moss enzymes can form dimers upon increasing protein concentration, setting them apart from the monomeric human and protozoal ADKs. Structural and kinetic analyses revealed a catalytically inactive unique dimer. Within the dimer, both active sites are mutually blocked. The activity of moss ADKs, exhibiting a higher propensity to dimerize, was 10-fold lower compared with maize ADKs. Two monomeric structures in a ternary complex highlight the characteristic transition from an open to a closed state upon substrate binding. This suggests that the oligomeric state switch can modulate the activity of moss ADKs and probably other plant ADKs. Moreover, dimer association represents a novel negative feedback mechanism, helping to maintain steady levels of adenosine and AMP.

• Klíčová slova
• Physcomitrella patens, Zea mays, Adenosine kinase, SnRK, crystal structure, cytokinin, overexpression, purine, riboside,
• MeSH
• adenosinkinasa * metabolismus   genetika   chemie   MeSH
• Arabidopsis genetika   enzymologie   metabolismus   MeSH
• kukuřice setá * enzymologie   genetika   metabolismus   MeSH
• mechy * enzymologie   genetika   metabolismus   MeSH
• multimerizace proteinu MeSH
• rostlinné proteiny * metabolismus   genetika   chemie   MeSH
• substrátová specifita MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• adenosinkinasa * MeSH
• rostlinné proteiny * MeSH

Photorhabdus laumondii is a well-known bacterium with a complex life cycle involving mutualism with nematodes of the genus Heterorhabditis and pathogenicity towards insect hosts. It provides an excellent model for studying the diverse roles of lectins, saccharide-binding proteins, in both symbiosis and pathogenicity. This study focuses on the seven-bladed β-propeller lectins of P. laumondii (PLLs), examining their biochemical properties (structure and saccharide specificity) and biological functions (gene expression, interactions with the nematode symbiont, and the host immune system response). Structural analyses revealed diverse oligomeric states among PLLs and a unique organisation of binding sites not described outside the PLL lectin family. Lectins exhibited high specificity for fucosylated and O-methylated saccharides with a significant avidity effect for multivalent ligands. Gene expression analysis across bacterial growth phases revealed that PLLs are predominantly expressed during the exponential phase. Interaction studies with the host immune system demonstrated that PLL5 uniquely induced melanisation in Galleria mellonella hemolymph. Furthermore, PLL2, PLL3, and PLL5 interfered with reactive oxygen species production in human blood cells, indicating their potential role in modulating host immune responses. Biofilm formation assays and binding studies with nematode life stages showed no significant involvement of PLLs in nematode colonization. Our findings highlight the primary role of PLLs in Photorhabdus pathogenicity rather than in symbiosis and offer valuable insight into the fascinating dynamics within the Photorhabdus-nematode-insect triparted system.

• Klíčová slova
• Photorhabdus, glycan array, lectin, nematode, structure-function study,
• MeSH
• bakteriální proteiny * chemie   metabolismus   genetika   MeSH
• hlístice * mikrobiologie   MeSH
• lektiny * chemie   metabolismus   genetika   MeSH
• lidé MeSH
• Photorhabdus * metabolismus   chemie   genetika   MeSH
• symbióza MeSH
• zvířata MeSH
• Check Tag
• lidé MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• bakteriální proteiny * MeSH
• lektiny * MeSH

S-adenosyl-l-methionine (SAM) is a crucial enzymatic cofactor that is conserved across all domains of life. Despite the pivotal role of this cofactor, its chirality at the sulfonium sulfur and the differing biological activities of its epimers, (S,S)-SAM and (R,S)-SAM, are often overlooked. Although enzymes predominantly utilize the (S,S)-SAM epimer, due to spontaneous epimerization at the sulfonium sulfur of SAM, the (R,S)-SAM epimer is present in all cells as well as in commercial SAM-containing products. Recently, an enzyme containing the DUF62 domain, identified as Salinispora tropica (StDUF62), has been shown to selectively hydrolyze (R,S)-SAM. It has been hypothesized that this function prevents the problematic accumulation of this epimer. Fluorinases, the only enzymes known to naturally incorporate fluorine into organic compounds, are homologous to enzymes of the DUF62 family. The discovery of unexpected diastereoselectivity of StDUF62 however raised an important question regarding the diastereoselectivity of the evolutionarily related bacterial fluorinase FlA1, an enzyme of significant importance. Given the relationship between these enzymes and their similar catalytic functions, it would be reasonable to hypothesize that FlA1 might also demonstrate activity toward the (R,S)-SAM diastereomer. Despite this homology, we report here the opposite diastereoselectivity of StDUF62 and Streptomyces sp. MA37 fluorinase (FlA1). The unusual lysine-free amino acid composition of StDUF62 suggests an evolutionary origin in haloadaptation; however, its SAM-hydrolyzing activity is greatly diminished at physiological concentrations of KCl or NaCl. We show that this inhibition is not caused solely by the competition with the chloride anion, as Na2SO4 at equivalent ionic strength is also greatly diminishing StDUF62 activity, contrary to the fluorinating activity of FlA1. Both adenosine and increased ionic strength promoted StDUF62 trimer formation, whereas increased ionic strength alone led to inhibition. Considering the contrast between the wasteful hydrolysis of (R,S)-SAM and the energetically efficient mechanisms of eukaryotic (R,S)-SAM recycling, we suggest that (R,S)-SAM hydrolysis might not be the physiological function of StDUF62.

• Publikační typ
• časopisecké články MeSH

Nedd4-2 E3 ligase regulates Na+ homeostasis by ubiquitinating various channels and membrane transporters, including the epithelial sodium channel ENaC. In turn, Nedd4-2 dysregulation leads to various conditions, including electrolytic imbalance, respiratory distress, hypertension, and kidney diseases. However, Nedd4-2 regulation remains mostly unclear. The present study aims at elucidating Nedd4-2 regulation by structurally characterizing Nedd4-2 and its complexes using several biophysical techniques. Our cryo-EM reconstruction shows that the C2 domain blocks the E2-binding surface of the HECT domain. This blockage, ubiquitin-binding exosite masking by the WW1 domain, catalytic C922 blockage and HECT domain stabilization provide the structural basis for Nedd4-2 autoinhibition. Furthermore, Ca2+-dependent C2 membrane binding disrupts C2/HECT interactions, but not Ca2+ alone, whereas 14-3-3 protein binds to a flexible region of Nedd4-2 containing the WW2 and WW3 domains, thereby inhibiting its catalytic activity and membrane binding. Overall, our data provide key mechanistic insights into Nedd4-2 regulation toward fostering the development of strategies targeting Nedd4-2 function.

• MeSH
• elektronová kryomikroskopie MeSH
• HEK293 buňky MeSH
• lidé MeSH
• molekulární modely MeSH
• proteinové domény MeSH
• proteiny 14-3-3 * metabolismus   chemie   MeSH
• ubikvitinace MeSH
• ubikvitinligasy Nedd4 * metabolismus   chemie   genetika   ultrastruktura   MeSH
• vápník * metabolismus   MeSH
• vazba proteinů MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• Nedd4 protein, human MeSH Prohlížeč
• Nedd4L protein, human MeSH Prohlížeč
• proteiny 14-3-3 * MeSH
• ubikvitinligasy Nedd4 * MeSH
• vápník * MeSH

The evolution of proteins is primarily driven by the combinatorial assembly of a limited set of pre-existing modules known as protein domains. This modular architecture not only supports the diversity of natural proteins but also provides a robust strategy for protein engineering, enabling the design of artificial proteins with enhanced or novel functions for various industrial applications. Among these functions, oligomerization plays a crucial role in enhancing protein activity, such as by increasing the binding capacity of antibodies. To investigate the potential of engineering oligomerization, we examined the transferability of the sequence domain encoded by exon 5 (Ex5), which was originally responsible for the oligomerization of ameloblastin (AMBN). We designed a two-domain protein composed of Ex5 in combination with a monomeric, globular, and highly stable protein, specifically calmodulin (CaM). CaM represents the opposite protein character to AMBN, which is highly disordered and has a dynamic character. This engineered protein, termed eCaM, successfully acquired an oligomeric function, inducing self-assembly under specific conditions. Biochemical and biophysical analyses revealed that the oligomerization of eCaM is both concentration- and time-dependent, with the process being reversible upon dilution. Furthermore, mutating a key oligomerization residue within Ex5 abolished the self-assembly of eCaM, confirming the essential role of the Ex5 motif in driving oligomerization. Our findings demonstrate that the oligomerization properties encoded by Ex5 can be effectively transferred to a new protein context, though the positioning of Ex5 within the protein structure is critical. This work highlights the potential of enhancing monomeric proteins with oligomeric functions, paving the way for industrial applications and the development of proteins with tailored properties.

• Publikační typ
• časopisecké články MeSH

In mycobacteria, σA is the primary sigma factor. This essential protein binds to RNA polymerase (RNAP) and mediates transcription initiation of housekeeping genes. Our knowledge about this factor in mycobacteria is limited. Here, we performed an unbiased search for interacting partners of Mycobacterium smegmatis σA. The search revealed a number of proteins; prominent among them was MoaB2. The σA-MoaB2 interaction was validated and characterized by several approaches, revealing that it likely does not require RNAP and is specific, as alternative σ factors (e.g., closely related σB) do not interact with MoaB2. The structure of MoaB2 was solved by X-ray crystallography. By immunoprecipitation and nuclear magnetic resonance, the unique, unstructured N-terminal domain of σA was identified to play a role in the σA-MoaB2 interaction. Functional experiments then showed that MoaB2 inhibits σA-dependent (but not σB-dependent) transcription and may increase the stability of σA in the cell. We propose that MoaB2, by sequestering σA, has a potential to modulate gene expression. In summary, this study has uncovered a new binding partner of mycobacterial σA, paving the way for future investigation of this phenomenon.IMPORTANCEMycobacteria cause serious human diseases such as tuberculosis and leprosy. The mycobacterial transcription machinery is unique, containing transcription factors such as RbpA, CarD, and the RNA polymerase (RNAP) core-interacting small RNA Ms1. Here, we extend our knowledge of the mycobacterial transcription apparatus by identifying MoaB2 as an interacting partner of σA, the primary sigma factor, and characterize its effects on transcription and σA stability. This information expands our knowledge of interacting partners of subunits of mycobacterial RNAP, providing opportunities for future development of antimycobacterial compounds.

• Klíčová slova
• MoaB2, RNA polymerase, mycobacteria, transcription, σA,
• MeSH
• bakteriální proteiny * metabolismus   genetika   MeSH
• DNA řízené RNA-polymerasy metabolismus   genetika   MeSH
• genetická transkripce MeSH
• krystalografie rentgenová MeSH
• Mycobacterium smegmatis * metabolismus   genetika   MeSH
• regulace genové exprese u bakterií * MeSH
• sigma faktor * metabolismus   genetika   MeSH
• transkripční faktory * metabolismus   genetika   MeSH
• vazba proteinů * MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• bakteriální proteiny * MeSH
• DNA řízené RNA-polymerasy MeSH
• sigma faktor * MeSH
• transkripční faktory * MeSH

Apoptosis signal-regulating kinase 1 (ASK1) is a crucial stress sensor, directing cells toward apoptosis, differentiation, and senescence via the p38 and JNK signaling pathways. ASK1 dysregulation has been associated with cancer and inflammatory, cardiovascular, and neurodegenerative diseases, among others. However, our limited knowledge of the underlying structural mechanism of ASK1 regulation hampers our ability to target this member of the MAP3K protein family towards developing therapeutic interventions for these disorders. Nevertheless, as a multidomain Ser/Thr protein kinase, ASK1 is regulated by a complex mechanism involving dimerization and interactions with several other proteins, including thioredoxin 1 (TRX1). Thus, the present study aims at structurally characterizing ASK1 and its complex with TRX1 using several biophysical techniques. As shown by cryo-EM analysis, in a state close to its active form, ASK1 is a compact and asymmetric dimer, which enables extensive interdomain and interchain interactions. These interactions stabilize the active conformation of the ASK1 kinase domain. In turn, TRX1 functions as a negative allosteric effector of ASK1, modifying the structure of the TRX1-binding domain and changing its interaction with the tetratricopeptide repeats domain. Consequently, TRX1 reduces access to the activation segment of the kinase domain. Overall, our findings not only clarify the role of ASK1 dimerization and inter-domain contacts but also provide key mechanistic insights into its regulation, thereby highlighting the potential of ASK1 protein-protein interactions as targets for anti-inflammatory therapy.

• Klíčová slova
• ASK1, MAP3K, MAPK signaling, biochemistry, chemical biology, human, molecular biophysics, protein kinase, structural biology, thioredoxin,
• MeSH
• apoptóza MeSH
• biofyzika MeSH
• elektronová kryomikroskopie MeSH
• MAP kinasa-kinasa-kinasa 5 * MeSH
• thioredoxiny * MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• MAP kinasa-kinasa-kinasa 5 * MeSH
• thioredoxiny * MeSH

Ca2+ /CaM-dependent protein kinase kinases 1 and 2 (CaMKK1 and CaMKK2) phosphorylate and enhance the catalytic activity of downstream kinases CaMKI, CaMKIV, and protein kinase B. Accordingly, CaMKK1 and CaMKK2 regulate key physiological and pathological processes, such as tumorigenesis, neuronal morphogenesis, synaptic plasticity, transcription factor activation, and cellular energy homeostasis, and promote cell survival. Both CaMKKs are partly inhibited by phosphorylation, which in turn triggers adaptor and scaffolding protein 14-3-3 binding. However, 14-3-3 binding only significantly affects CaMKK1 function. CaMKK2 activity remains almost unchanged after complex formation for reasons still unclear. Here, we aim at structurally characterizing CaMKK1:14-3-3 and CaMKK2:14-3-3 complexes by SAXS, H/D exchange coupled to MS, and fluorescence spectroscopy. The results revealed that complex formation suppresses the interaction of both phosphorylated CaMKKs with Ca2+ /CaM and affects the structure of their kinase domains and autoinhibitory segments. But these effects are much stronger on CaMKK1 than on CaMKK2 because the CaMKK1:14-3-3γ complex has a more compact and rigid structure in which the active site of the kinase domain directly interacts with the last two C-terminal helices of the 14-3-3γ protein, thereby inhibiting CaMKK1. In contrast, the CaMKK2:14-3-3 complex has a looser and more flexible structure, so 14-3-3 binding only negligibly affects the catalytic activity of CaMKK2. Therefore, Ca2+ /CaM binding suppression and the interaction of the kinase active site of CaMKK1 with the last two C-terminal helices of 14-3-3γ protein provide the structural basis for 14-3-3-mediated CaMKK1 inhibition.

• Klíčová slova
• 14-3-3 proteins, CaMKK, SAXS, calcium/calmodulin-dependent protein kinase, fluorescence spectroscopy, hydrogen/deuterium exchange coupled to MS, protein-protein interaction,
• MeSH
• difrakce rentgenového záření MeSH
• fosforylace MeSH
• katalytická doména MeSH
• kinasa proteinkinasy závislé na vápníku a kalmodulinu * chemie   metabolismus   MeSH
• maloúhlový rozptyl MeSH
• proteiny 14-3-3 * metabolismus   MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• kinasa proteinkinasy závislé na vápníku a kalmodulinu * MeSH
• proteiny 14-3-3 * MeSH

The physical stability of peptide-based drugs is of great interest to the pharmaceutical industry. Glucagon-like peptide 1 (GLP-1) is a 31-amino acid peptide hormone, the analogs of which are frequently used in the treatment of type 2 diabetes. We investigated the physical stability of GLP-1 and its C-terminal amide derivative, GLP-1-Am, both of which aggregate into amyloid fibrils. While off-pathway oligomers have been proposed to explain the unusual aggregation kinetics observed previously for GLP-1 under specific conditions, these oligomers have not been studied in any detail. Such states are important as they may represent potential sources of cytotoxicity and immunogenicity. Here, we identified and isolated stable, low-molecular-weight oligomers of GLP-1 and GLP-1-Am, using size-exclusion chromatography. Under the conditions studied, isolated oligomers were shown to be resistant to fibrillation or dissociation. These oligomers contain between two and five polypeptide chains and they have a highly disordered structure as indicated by a variety of spectroscopic techniques. They are highly stable with respect to time, temperature, or agitation despite their noncovalent character, which was established using liquid chromatography-mass spectrometry and sodium dodecyl sulfate-polyacrylamide gel electrophoresis. These results provide evidence of stable, low-molecular-weight oligomers that are formed by an off-pathway mechanism which competes with amyloid fibril formation.

• Klíčová slova
• aggregation, amyloid, glucagon-like peptide 1, oligomers, self-assembly,
• MeSH
• amyloid chemie   MeSH
• amyloidní beta-protein chemie   MeSH
• diabetes mellitus 2. typu * MeSH
• gelová chromatografie MeSH
• glukagonu podobný peptid 1 * MeSH
• lidé MeSH
• peptidové fragmenty chemie   MeSH
• peptidy MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• amyloid MeSH
• amyloidní beta-protein MeSH
• glukagonu podobný peptid 1 * MeSH
• peptidové fragmenty MeSH
• peptidy MeSH

BACKGROUND: Apolipoprotein E (ApoE) ε4 genotype is the most prevalent risk factor for late-onset Alzheimer's Disease (AD). Although ApoE4 differs from its non-pathological ApoE3 isoform only by the C112R mutation, the molecular mechanism of its proteinopathy is unknown. METHODS: Here, we reveal the molecular mechanism of ApoE4 aggregation using a combination of experimental and computational techniques, including X-ray crystallography, site-directed mutagenesis, hydrogen-deuterium mass spectrometry (HDX-MS), static light scattering and molecular dynamics simulations. Treatment of ApoE ε3/ε3 and ε4/ε4 cerebral organoids with tramiprosate was used to compare the effect of tramiprosate on ApoE4 aggregation at the cellular level. RESULTS: We found that C112R substitution in ApoE4 induces long-distance (> 15 Å) conformational changes leading to the formation of a V-shaped dimeric unit that is geometrically different and more aggregation-prone than the ApoE3 structure. AD drug candidate tramiprosate and its metabolite 3-sulfopropanoic acid induce ApoE3-like conformational behavior in ApoE4 and reduce its aggregation propensity. Analysis of ApoE ε4/ε4 cerebral organoids treated with tramiprosate revealed its effect on cholesteryl esters, the storage products of excess cholesterol. CONCLUSIONS: Our results connect the ApoE4 structure with its aggregation propensity, providing a new druggable target for neurodegeneration and ageing.

• Klíčová slova
• 3-sulfopropanoic acid, Aggregation, Alzheimer’s disease, Apolipoprotein E, Cerebral organoids, HDX-MS, Lipidomics, Molecular dynamics, Neurodegeneration, Protein crystallography, Proteomics, Tramiprosate,
• MeSH
• Alzheimerova nemoc * farmakoterapie   genetika   metabolismus   MeSH
• apolipoprotein E3 genetika   MeSH
• apolipoprotein E4 * genetika   metabolismus   MeSH
• apolipoproteiny E genetika   MeSH
• lidé MeSH
• mutace genetika   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• apolipoprotein E3 MeSH
• apolipoprotein E4 * MeSH
• apolipoproteiny E MeSH
• tramiprosate MeSH Prohlížeč