Molecular insights into the regulatory landscape of PKC-related kinase-2 (PRK2/PKN2) using targeted small compounds.

The PKC-related kinases (PRKs, also termed PKNs) are important in cell migration, cancer, hepatitis C infection, and nutrient sensing. They belong to a group of protein kinases called AGC kinases that share common features like a C-terminal extension to the catalytic domain comprising a hydrophobic motif. PRKs are regulated by N-terminal domains, a pseudosubstrate sequence, Rho-binding domains, and a C2 domain involved in inhibition and dimerization, while Rho and lipids are activators. We investigated the allosteric regulation of PRK2 and its interaction with its upstream kinase PDK1 using a chemical biology approach. We confirmed the phosphoinositide-dependent protein kinase 1 (PDK1)-interacting fragment (PIF)-mediated docking interaction of PRK2 with PDK1 and showed that this interaction can be modulated allosterically. We showed that the polypeptide PIFtide and a small compound binding to the PIF-pocket of PRK2 were allosteric activators, by displacing the pseudosubstrate PKL region from the active site. In addition, a small compound binding to the PIF-pocket allosterically inhibited the catalytic activity of PRK2. Together, we confirmed the docking interaction and allostery between PRK2 and PDK1 and described an allosteric communication between the PIF-pocket and the active site of PRK2, both modulating the conformation of the ATP-binding site and the pseudosubstrate PKL-binding site. Our study highlights the allosteric modulation of the activity and the conformation of PRK2 in addition to the existence of at least two different complexes between PRK2 and its upstream kinase PDK1. Finally, the study highlights the potential for developing allosteric drugs to modulate PRK2 kinase conformations and catalytic activity.

Discovery of a Potent Dual SLK/STK10 Inhibitor Based on a Maleimide Scaffold.

SLK (STE20-like kinase) and STK10 (serine/threonine kinase 10) are closely related kinases whose enzymatic activity is linked to the regulation of ezrin, radixin, and moesin function and to the regulation of lymphocyte migration and the cell cycle. We identified a series of 3-anilino-4-arylmaleimides as dual inhibitors of SLK and STK10 with good kinome-wide selectivity. Optimization of this series led to multiple SLK/STK10 inhibitors with nanomolar potency. Crystal structures of exemplar inhibitors bound to SLK and STK10 demonstrated the binding mode of the inhibitors and rationalized their selectivity. Cellular target engagement assays demonstrated the binding of the inhibitors to SLK and STK10 in cells. Further selectivity analyses, including analysis of activity of the reported inhibitors against off-targets in cells, identified compound 31 as the most potent and selective inhibitor of SLK and STK10 yet reported.

An Ancestry Perspective of the Evolution of PBS1 Proteins in Plants.

The AVRPPHB SUSCEPTIBLE1 (PBS1) and RESISTANCE TO PSEUDOMONAS SYRINGAE 5 (RPS5) proteins are involved in signal transduction to evoke innate plant immune response. In Arabidopsis, PBS1 is cleaved by the AvrPphB (Pseudomonas phaseolicola Avirulence protein B) protease, activating RPS5 and turning in a hypersensitive response (HR). We searched for PBS1 orthologs to trace their origin and evolution. PBS1 orthologs were found in embryophytes and in other plant taxa but with lower similarity. PBS1 phylogenetic analysis indicates high divergence, suggesting that the decoy function described for Arabidopsis PBS1 might be associated with a small fraction of orthologs. Ancestral reconstruction analysis suggests an elevated diversity in the amino acid sequence within the described motifs. All the orthologs contain the conserved PBS1 kinase subdomains, whereas the cleavage motif is present in several embryophyte orthologs but absent in most other taxa. The putative resistance recognition motifs in PBS1 orthologs are highly diverse. PBS1 cleavage site motif is exposed in some 3D structure predictions, whereas it is not in others, suggesting different modes of regulation and functions in PBS1 orthologs. Our findings suggest that PBS1 originated in the lineage that gave rise to embryophytes, with the angiosperm sequences forming a separate clade from pteridophyte proteins.

Trib1 regulates T cell differentiation during chronic infection by restraining the effector program.

In chronic infections, the immune response fails to control virus, leading to persistent antigen stimulation and the progressive development of T cell exhaustion. T cell effector differentiation is poorly understood in the context of exhaustion, but targeting effector programs may provide new strategies for reinvigorating T cell function. We identified Tribbles pseudokinase 1 (Trib1) as a central regulator of antiviral T cell immunity, where loss of Trib1 led to a sustained enrichment of effector-like KLRG1+ T cells, enhanced function, and improved viral control. Single-cell profiling revealed that Trib1 restrains a population of KLRG1+ effector CD8 T cells that is transcriptionally distinct from exhausted cells. Mechanistically, we identified an interaction between Trib1 and the T cell receptor (TCR) signaling activator, MALT1, which disrupted MALT1 signaling complexes. These data identify Trib1 as a negative regulator of TCR signaling and downstream function, and reveal a link between Trib1 and effector versus exhausted T cell differentiation that can be targeted to improve antiviral immunity.

Biological Activities and Molecular Docking of Brassinosteroids 24-Norcholane Type Analogs.

The quest and design of new brassinosteroids analogs is a matter of current interest. Herein, the effect of short alkyl side chains and the configuration at C22 on the growth-promoting activity of a series of new brassinosteroid 24-norcholan-type analogs have been evaluated by the rice leaf inclination test using brassinolide as positive control. The highest activities were found for triol 3 with a C22(S) configuration and monobenzoylated derivatives. A docking study of these compounds into the active site of the Brassinosteroid Insensitive 1(BRI1)-ligand-BRI1-Associated Receptor Kinase 1 (BAK1) complex was performed using AutoDock Vina, and protein-ligand contacts were analyzed using LigPlot+. The results suggest that the hydrophobic interactions of ligands with the receptor BRI1LRR and hydrogen bonding with BAK1 in the complex are important for ligand recognition. For monobenzoylated derivatives, the absence of the hydrophobic end in the alkyl chain seems to be compensated by the benzoyl group. Thus, it would be interesting to determine if this result depends on the nature of the substituent group. Finally, mixtures of S/R triols 3/4 exhibit activities that are comparable or even better than those found for brassinolide. Thus, these compounds are potential candidates for application in agriculture to improve the growth and yield of plants against various types of biotic and abiotic stress.

Insights into the full-length SRPK2 structure and its hydrodynamic behavior.

The serine/arginine-rich protein kinase 2 (SRPK2) has been reported as upregulated in several cancer types, with roles in hallmarks such as cell migration, growth, and apoptosis. These findings have indicated that SRPK2 is a promising emerging target in drug discovery initiatives. Although high-resolution models are available for SRPK2 (PDB 2X7G), they have been obtained with a heavily truncated recombinant protein version (~50% of the primary structure), due to the presence of long intrinsically unstructured regions. In the present work, we sought to characterize the structure of a full-length recombinant version of SRPK2 in solution. Low-resolution Small-Angle X-ray Scattering data were obtained for both versions of SRPK2. The truncated ΔNΔS-SRPK2 presented a propensity to dimerize at higher concentrations whereas the full-length SRPK2 was mainly found as dimers. The hydrodynamic behavior of the full-length SRPK2 was further investigated by analytical size exclusion chromatography and sedimentation velocity analytical ultracentrifugation experiments. SRPK2 behaved as a monomer-dimer equilibrium and both forms have an elongated shape in solution, pointing to a stretched-to-closed tendency among the conformational plasticity observed. Taken together, these findings allowed us to define unique structural features of the SRPK2 within SRPK family, characterized by its flexible regions outside the bipartite kinase domain.

The prp4 kinase gene and related spliceosome factor genes in Trichophyton rubrum respond to nutrients and antifungals.

PURPOSE: Trichophyton rubrum is a dermatophyte that causes most human superficial mycoses worldwide. The spliceosome, a large ribonucleoprotein complex responsible for pre-mRNA processing, may confer adaptive advantages to deal with different stresses. Here, we assessed the structural aspects of the Prp4 kinase protein and other pre-mRNA-splicing factors (Prps) in T. rubrum grown in different protein sources and exposed to antifungal drugs. METHODOLOGY: Quantitative Reverse Transcription PCR (RT-PCR) assessed the modulation of prp1, prp31, prp8 and prp4 kinase genes after exposure of T. rubrum to sub-lethal doses of amphotericin B, caspofungin and acriflavine, or after T. rubrum growth on keratin sources for 48 and 72 h. We also performed the in silico analysis of the domain organization of Prps orthologues from filamentous fungi and yeasts. RESULTS: The prp4 gene was modulated in a time-dependent manner. Transcription levels were mostly up-regulated when T. rubrum was grown on keratin for 72 h, while exposure to amphotericin B promoted prp4 gene down-regulation at the same time point. We also observed co-expression of prp1 and prp31, and their down-regulation after amphotericin B exposure. In silico analysis revealed a conserved domain organization for most Prps orthologues with slight differences, which were mostly related to structural elements such as repetition domains in Prp1 and complexity in motif assembly for the Prp4 kinase. These differences were mainly observed in dermatophyte species and may alter protein interactions and substrate affinity. CONCLUSION: Our results improve the understanding of spliceosome proteins in fungi as well as their roles in adaptation to different environmental situations.

Mycobacterium tuberculosis serine/threonine protein kinases: structural information for the design of their specific ATP-competitive inhibitors.

In the last decades, human protein kinases (PKs) have been relevant as targets in the development of novel therapies against many diseases, but the study of Mycobacterium tuberculosis PKs (MTPKs) involved in tuberculosis pathogenesis began much later and has not yet reached an advanced stage of development. To increase knowledge of these enzymes, in this work we studied the structural features of MTPKs, with focus on their ATP-binding sites and their interactions with inhibitors. PknA, PknB, and PknG are the most studied MTPKs, which were previously crystallized; ATP-competitive inhibitors have been designed against them in the last decade. In the current work, reported PknA, PknB, and PknG inhibitors were extracted from literature and their orientations inside the ATP-binding site were proposed by using docking method. With this information, interaction fingerprints were elaborated, which reveal the more relevant residues for establishing chemical interactions with inhibitors. The non-crystallized MTPKs PknD, PknF, PknH, PknJ, PknK, and PknL were also studied; their three-dimensional structural models were developed by using homology modeling. The main characteristics of MTPK ATP-binding sites (the non-crystallized and crystallized MTPKs, including PknE and PknI) were accounted; schemes of the main polar and nonpolar groups inside their ATP-binding sites were constructed, which are suitable for a major understanding of these proteins as antituberculotic targets. These schemes could be used for establishing comparisons between MTPKs and human PKs in order to increase selectivity of MTPK inhibitors. As a key tool for guiding medicinal chemists interested in the design of novel MTPK inhibitors, our work provides a map of the structural elements relevant for the design of more selective ATP-competitive MTPK inhibitors.

Crosstalk between the serine/threonine kinase StkP and the response regulator ComE controls the stress response and intracellular survival of Streptococcus pneumoniae.

Streptococcus pneumoniae is an opportunistic human bacterial pathogen that usually colonizes the upper respiratory tract, but the invasion and survival mechanism in respiratory epithelial cells remains elusive. Previously, we described that acidic stress-induced lysis (ASIL) and intracellular survival are controlled by ComE through a yet unknown activation mechanism under acidic conditions, which is independent of the ComD histidine kinase that activates this response regulator for competence development at pH 7.8. Here, we demonstrate that the serine/threonine kinase StkP is essential for ASIL, and show that StkP phosphorylates ComE at Thr128. Molecular dynamic simulations predicted that Thr128-phosphorylation induces conformational changes on ComE's DNA-binding domain. Using nonphosphorylatable (ComET128A) and phosphomimetic (ComET128E) proteins, we confirmed that Thr128-phosphorylation increased the DNA-binding affinity of ComE. The non-phosphorylated form of ComE interacted more strongly with StkP than the phosphomimetic form at acidic pH, suggesting that pH facilitated crosstalk. To identify the ComE-regulated genes under acidic conditions, a comparative transcriptomic analysis was performed between the comET128A and wt strains, and differential expression of 104 genes involved in different cellular processes was detected, suggesting that the StkP/ComE pathway induced global changes in response to acidic stress. In the comET128A mutant, the repression of spxB and sodA correlated with decreased H2O2 production, whereas the reduced expression of murN correlated with an increased resistance to cell wall antibiotic-induced lysis, compatible with cell wall alterations. In the comET128A mutant, ASIL was blocked and acid tolerance response was higher compared to the wt strain. These phenotypes, accompanied with low H2O2 production, are likely responsible for the increased survival in pneumocytes of the comET128A mutant. We propose that the StkP/ComE pathway controls the stress response, thus affecting the intracellular survival of S. pneumoniae in pneumocytes, one of the first barriers that this pathogen must cross to establish an infection.

C-terminally truncated, kidney-specific variants of the WNK4 kinase lack several sites that regulate its activity.

WNK lysine-deficient protein kinase 4 (WNK4) is an important regulator of renal salt handling. Mutations in its gene cause pseudohypoaldosteronism type II, mainly arising from overactivation of the renal Na+/Cl- cotransporter (NCC). In addition to full-length WNK4, we have observed faster migrating bands (between 95 and 130 kDa) in Western blots of kidney lysates. Therefore, we hypothesized that these could correspond to uncharacterized WNK4 variants. Here, using several WNK4 antibodies and WNK4-/- mice as controls, we showed that these bands indeed correspond to short WNK4 variants that are not observed in other tissue lysates. LC-MS/MS confirmed these bands as WNK4 variants that lack C-terminal segments. In HEK293 cells, truncation of WNK4's C terminus at several positions increased its kinase activity toward Ste20-related proline/alanine-rich kinase (SPAK), unless the truncated segment included the SPAK-binding site. Of note, this gain-of-function effect was due to the loss of a protein phosphatase 1 (PP1)-binding site in WNK4. Cotransfection with PP1 resulted in WNK4 dephosphorylation, an activity that was abrogated in the PP1-binding site WNK4 mutant. The electrophoretic mobility of the in vivo short variants of renal WNK4 suggested that they lack the SPAK-binding site and thus may not behave as constitutively active kinases toward SPAK. Finally, we show that at least one of the WNK4 short variants may be produced by proteolysis involving a Zn2+-dependent metalloprotease, as recombinant full-length WNK4 was cleaved when incubated with kidney lysate.

Phosphorylation of IRE1 at S729 regulates RIDD in B cells and antibody production after immunization.

To relieve endoplasmic reticulum (ER) stress, IRE1 splices XBP1 messenger RNA (mRNA) or engages regulated IRE1-dependent decay (RIDD) of other mRNAs. Upon XBP1 deficiency, IRE1 switches to perform RIDD. We examined IRE1 in XBP1-deficient B cells and discovered that IRE1 undergoes phosphorylation at S729. We generated an anti-phospho-S729 antibody to investigate such phosphorylation. Compared with pharmacological ER stress inducers or Toll-like receptor ligands, the bacterial subtilase cytotoxin has an unusual capability in causing rapid and strong phosphorylation at S729 and triggering B cells to express spliced XBP1. To assess the function of S729 in IRE1, we generated S729A knock-in mice and found S729 is critically important for lipopolysaccharide-stimulated plasmablasts to respond to additional ER stress and for antibody production in response to immunization. We further crossed mice carrying an S729A mutation or ΔIRE1 (missing the kinase domain) with B cell-specific XBP1-deficient mice to trigger RIDD and discovered a critical role for S729 in regulating RIDD in B cells.

Structure and function of Per-ARNT-Sim domains and their possible role in the life-cycle biology of Trypanosoma cruzi.

Per-ARNT-Sim (PAS) domains of proteins play important roles as modules for signalling and cellular regulation processes in widely diverse organisms such as Archaea, Bacteria, protists, plants, yeasts, insects and vertebrates. These domains are present in many proteins where they are used as sensors of stimuli and modules for protein interactions. Characteristically, they can bind a broad spectrum of molecules. Such binding causes the domain to trigger a specific cellular response or to make the protein containing the domain susceptible to responding to additional physical or chemical signals. Different PAS proteins have the ability to sense redox potential, light, oxygen, energy levels, carboxylic acids, fatty acids and several other stimuli. Such proteins have been found to be involved in cellular processes such as development, virulence, sporulation, adaptation to hypoxia, circadian cycle, metabolism and gene regulation and expression. Our analysis of the genome of different kinetoplastid species revealed the presence of PAS domains also in different predicted kinases from these protists. Open-reading frames coding for these PAS-kinases are unusually large. In addition, the products of these genes appear to contain in their structure combinations of domains uncommon in other eukaryotes. The physiological significance of PAS domains in these parasites, specifically in Trypanosoma cruzi, is discussed.

Structural characterization of human Vaccinia-Related Kinases (VRK) bound to small-molecule inhibitors identifies different P-loop conformations.

The human genome encodes two active Vaccinia-related protein kinases (VRK), VRK1 and VRK2. These proteins have been implicated in a number of cellular processes and linked to a variety of tumors. However, understanding the cellular role of VRKs and establishing their potential use as targets for therapeutic intervention has been limited by the lack of tool compounds that can specifically modulate the activity of these kinases in cells. Here we identified BI-D1870, a dihydropteridine inhibitor of RSK kinases, as a promising starting point for the development of chemical probes targeting the active VRKs. We solved co-crystal structures of both VRK1 and VRK2 bound to BI-D1870 and of VRK1 bound to two broad-spectrum inhibitors. These structures revealed that both VRKs can adopt a P-loop folded conformation, which is stabilized by different mechanisms on each protein. Based on these structures, we suggest modifications to the dihydropteridine scaffold that can be explored to produce potent and specific inhibitors towards VRK1 and VRK2.

Molecular characterization of the gene checkpoint homolog 1 in Daphnia carinata during different reproductive phases.

Full-length cDNA of the gene checkpoint homolog 1 (Chk1) was cloned from Daphnia carinata and designated DcarChk1. DcarChk1 cDNA was 1817 bp in length and encoded a 497-amino acid polypeptide. Phylogenetic analyses revealed that DcarChk1 was most closely related to Chk1 of Daphnia pulex, followed by homologous genes of insects. Expression of DcarChk1 was higher in adult Daphnia than in larvae, and significantly higher in males than females, as determined by real-time polymerase chain reaction analysis. Using whole-mount in situ hybridization techniques, DcarChk1 in parthenogenetic females was found to be expressed mainly on the head surface, capillus, and carapace valve edge. In contrast, in sexual females, DcarChk1 was expressed mainly in the joint of the second antenna, and in the thoracic limbs and capillus. These results suggest that DcarChk1 plays a significant role in both the growth and development, as well as in regulating reproductive plasticity, in D. carinata.

In silico studies on the interaction between bioactive ligands and ALK5, a biological target related to the cancer treatment.

Studies have showed that there are many biological targets related to the cancer treatment, for example, TGF type I receptor (TGF-ßRI or ALK5). The ALK5 inhibition is a strategy to treat some types of cancer, such as breast, lung, and pancreas. Here, we performed CoMFA and CoMSIA studies for 70 ligands with ALK5 inhibition. The internal validation for both models (q(2)LOO = 0.887 and 0.822, respectively) showed their robustness, while the external validations showed their predictive power (CoMFA: r(2)test = 0.998; CoMSIA: r(2)test = 0.975). After all validations, CoMFA and CoMSIA maps indicated physicochemical evidences on the main factors involved in the interaction between bioactive ligands and ALK5. Therefore, these results suggest molecular modifications to design new ALK5 inhibitors.

Interactome analysis and design of inhibitors against selected protein targets of Ser/Thr protein kinase (STPK) signaling pathways in Mycobacterium tuberculosis H37Rv.

Tuberculosis continues to be a major cause of mortality worldwide despite significant advances in chemotherapy and development of the BCG vaccine. Although curable, the tuberculosis treatment period (6-9 months) presents many concerns, including patient noncompliance and the development of drug toxicity and drug resistance. This study aimed to understand the protein-protein interactions of key proteins involved in the Mycobacterium tuberculosis STPK signal transduction pathway (such as PknB, PknE, and PstP); in addition, we attempted to identify promising leads for the inhibition of protein-protein interactions. Interactome analyses revealed the interactions of these protein targets with several other proteins, including PknG and PbpA. Drug-like candidates were screened based on Lipinski's rule of five and the absorption digestion metabolism excretion toxicity. Molecular docking of the target proteins with the selected ligands identified cryptolepine HCl to be a common molecule interacting with all protein targets (with a good docking score). The generation of a pharmacophore model for cryptolepine HCl revealed three pharmacophoric regions: aromatic hydrocarbon, hydrogen bond acceptor, and hydrogen bond donor, which play important roles in its interaction with the protein targets. Therefore, cryptolepine HCl appears to be a promising drug candidate for further optimization and validation against M. tuberculosis.

Potential Antileukemia Effect and Structural Analyses of SRPK Inhibition by N-(2-(Piperidin-1-yl)-5-(Trifluoromethyl)Phenyl)Isonicotinamide (SRPIN340).

Dysregulation of pre-mRNA splicing machinery activity has been related to the biogenesis of several diseases. The serine/arginine-rich protein kinase family (SRPKs) plays a critical role in regulating pre-mRNA splicing events through the extensive phosphorylation of splicing factors from the family of serine/arginine-rich proteins (SR proteins). Previous investigations have described the overexpression of SRPK1 and SRPK2 in leukemia and other cancer types, suggesting that they would be useful targets for developing novel antitumor strategies. Herein, we evaluated the effect of selective pharmacological SRPK inhibition by N-(2-(piperidin-1-yl)-5-(trifluoromethyl)phenyl)isonicotinamide (SRPIN340) on the viability of lymphoid and myeloid leukemia cell lines. Along with significant cytotoxic activity, the effect of treatments in regulating the phosphorylation of the SR protein family and in altering the expression of MAP2K1, MAP2K2, VEGF and FAS genes were also assessed. Furthermore, we found that pharmacological inhibition of SRPKs can trigger early and late events of apoptosis. Finally, intrinsic tryptophan fluorescence emission, molecular docking and molecular dynamics were analyzed to gain structural information on the SRPK/SRPIN340 complex. These data suggest that SRPK pharmacological inhibition should be considered as an alternative therapeutic strategy for fighting leukemias. Moreover, the obtained SRPK-ligand interaction data provide useful structural information to guide further medicinal chemistry efforts towards the development of novel drug candidates.

Characterization of the human NEK7 interactome suggests catalytic and regulatory properties distinct from those of NEK6.

Human NEK7 is a regulator of cell division and plays an important role in growth and survival of mammalian cells. Human NEK6 and NEK7 are closely related, consisting of a conserved C-terminal catalytic domain and a nonconserved and disordered N-terminal regulatory domain, crucial to mediate the interactions with their respective proteins. Here, in order to better understand NEK7 cellular functions, we characterize the NEK7 interactome by two screening approaches: one using a yeast two-hybrid system and the other based on immunoprecipitation followed by mass spectrometry analysis. These approaches led to the identification of 61 NEK7 interactors that contribute to a variety of biological processes, including cell division. Combining additional interaction and phosphorylation assays from yeast two-hybrid screens, we validated CC2D1A, TUBB2B, MNAT1, and NEK9 proteins as potential NEK7 interactors and substrates. Notably, endogenous RGS2, TUBB, MNAT1, NEK9, and PLEKHA8 localized with NEK7 at key sites throughout the cell cycle, especially during mitosis and cytokinesis. Furthermore, we obtained evidence that the closely related kinases NEK6 and NEK7 do not share common interactors, with the exception of NEK9, and display different modes of protein interaction, depending on their N- and C-terminal regions, in distinct fashions. In summary, our work shows for the first time a comprehensive NEK7 interactome that, combined with functional in vitro and in vivo assays, suggests that NEK7 is a multifunctional kinase acting in different cellular processes in concert with cell division signaling and independently of NEK6.

Structural and phylogenetic analysis of Pto-type disease resistance gene candidates in Hevea brasiliensis.

The tomato Pto gene encodes a serine/threonine kinase (STK) whose molecular characterization has provided valuable insights into the disease resistance mechanism of tomato. Therefore, Pto is considered as a promising candidate for engineering broad-spectrum pathogen resistance in this crop. In this study, a pair of degenerate primers based on conserved subdomains of plant STKs similar to the tomato Pto protein was used to amplify similar sequences in a hevea cultivar (Hevea brasiliensis Muell. Arg). A fragment of ~550 bp was amplified, cloned, and sequenced. The sequence analysis of several clones revealed 12 distinct sequences highly similar to STKs. Based on their significant similarity with the tomato Pto protein (BLASTX E value<3e-53), seven sequences were classified as Pto resistance gene candidates (Pto-RGCs). Multiple sequence alignment of the hevea Pto-RGC products revealed that these sequences contain several conserved subdomains present in most STKs, as well as several conserved residues that are crucial for Pto function. Moreover, phylogenetic analysis showed that the hevea Pto-RGCs clustered with Pto, suggesting a common evolutionary origin with this resistance gene. The Pto-RGCs isolated in this study represent a valuable sequence resource that could assist in the development of disease resistance in hevea.

Inhibition of Mycobacterium tuberculosis PknG by non-catalytic rubredoxin domain specific modification: reaction of an electrophilic nitro-fatty acid with the Fe-S center.

PknG from Mycobacterium tuberculosis is a Ser/Thr protein kinase that regulates key metabolic processes within the bacterial cell as well as signaling pathways from the infected host cell. This multidomain protein has a conserved canonical kinase domain with N- and C-terminal flanking regions of unclear functional roles. The N-terminus harbors a rubredoxin-like domain (Rbx), a bacterial protein module characterized by an iron ion coordinated by four cysteine residues. Disruption of the Rbx-metal binding site by simultaneous mutations of all the key cysteine residues significantly impairs PknG activity. This encouraged us to evaluate the effect of a nitro-fatty acid (9- and 10-nitro-octadeca-9-cis-enoic acid; OA-NO2) on PknG activity. Fatty acid nitroalkenes are electrophilic species produced during inflammation and metabolism that react with nucleophilic residues of target proteins (i.e., Cys and His), modulating protein function and subcellular distribution in a reversible manner. Here, we show that OA-NO2 inhibits kinase activity by covalently adducting PknG remote from the catalytic domain. Mass spectrometry-based analysis established that cysteines located at Rbx are the specific targets of the nitroalkene. Cys-nitroalkylation is a Michael addition reaction typically reverted by thiols. However, the reversible OA-NO2-mediated nitroalkylation of the kinase results in an irreversible inhibition of PknG. Cys adduction by OA-NO2 induced iron release from the Rbx domain, revealing a new strategy for the specific inhibition of PknG. These results affirm the relevance of the Rbx domain as a target for PknG inhibition and support that electrophilic lipid reactions of Rbx-Cys may represent a new drug strategy for specific PknG inhibition.