C-terminal Domain Research Articles

Systemic antibody responses against gut microbiota flagellins implicate shared and divergent immune reactivity in Crohn’s disease and chronic fatigue syndrome

BackgroundElevated systemic antibody responses against gut microbiota flagellins are observed in both Crohn’s disease (CD) and myalgic encephalomyelitis/chronic fatigue syndrome (ME/CFS), suggesting potential serological biomarkers for diagnosis. However, flagellin-specific antibody repertoires and functional roles in the diseases remain incompletely understood. Bacterial flagellins can be categorized into three types depending on their interaction with toll-like receptor 5 (TLR5): (1) “stimulator” and (2) “silent” flagellins, which bind TLR5 through a conserved N-terminal motif, with only stimulators activating TLR5 (involving a C-terminal domain); (3) “evader” flagellins of pathogens, which entirely circumvent TLR5 activation via mutations in the N-terminal TLR5 binding motif.ResultsHere, we show that both CD and ME/CFS patients exhibit elevated antibody responses against distinct regions of flagellins compared to healthy individuals. N-terminal binding to Lachnospiraceae flagellins was comparable in both diseases, while C-terminal binding was more prevalent in CD. N-terminal antibody-bound flagellin sequences were similar across CD and ME/CFS, resembling “stimulator” and “silent” flagellins more than evaders. However, C-terminal antibody-bound flagellins showed a higher resemblance to the stimulator than to silent flagellins in CD, which was not observed in ME/CFS.ConclusionsThese findings suggest that antibody binding to the N-terminal domain of stimulator and silent flagellins may impact TLR5 activation in both CD and ME/CFS patients. Blocking this interaction could lead commensal bacteria to be recognized as pathogenic evaders, potentially contributing to dysregulation in both diseases. Furthermore, elevated antibody binding to the C-terminal domain of stimulator flagellins in CD may explain pathophysiological differences between the diseases. Overall, these results highlight the diagnostic potential of these antibody responses and lay a foundation for deeper mechanistic studies of flagellin/TLR5 interactions and their impact on innate/adaptive immunity balance.Graphical 3dk6-s1stA6fneys_e7fgXVideo

TDP-43 Promotes Amyloid-Beta Toxicity by Delaying Fibril Maturation via Direct Molecular Interaction.

Amyloid-β (Aβ) is a peptide that undergoes self-assembly into amyloid fibrils, which compose the hallmark plaques observed in Alzheimer's disease (AD). TAR DNA-binding protein 43 (TDP-43) is a protein with mislocalization and aggregation implicated in amyotrophic lateral sclerosis and other neurodegenerative diseases. Recent work suggests that TDP-43 may interact with Aβ, inhibiting the formation of amyloid fibrils and worsening AD pathology, but the molecular details of their interaction remain unknown. Using all-atom discrete molecular dynamics simulations, we systematically investigated the direct molecular interaction between Aβ and TDP-43. We found that Aβ monomers were able to bind near the flexible nuclear localization sequence of the N-terminal domain (NTD) of TDP-43, adopting β-sheet rich conformations that were promoted by the interaction. Furthermore, Aβ associated with the nucleic acid binding interface of the tandem RNA recognition motifs of TDP-43 via electrostatic interactions. Using the computational peptide array method, we found the strongest C-terminal domain interaction with Aβ to be within the amyloidogenic core region of TDP-43. With experimental evidence suggesting that the NTD is necessary for inhibiting Aβ fibril growth, we also simulated the NTD with an Aβ40 fibril seed. We found that the NTD was able to strongly bind the elongation surface of the fibril seed via extensive hydrogen bonding and could also diffuse along the lateral surface via electrostatic interactions. Our results suggest that TDP-43 binding to the elongation surface, thereby sterically blocking Aβ monomer addition, is responsible for the experimentally observed inhibition of fibril growth. We conclude that TDP-43 may promote Aβ toxicity by stabilizing the oligomeric state and kinetically delaying fibril maturation.

Investigating Medin Cleavage Accessibility in MfgE8: Conformational Insights Derived from Molecular Dynamics Simulations and AlphaFold2 Models.

Recent studies have indicated that the human amyloidogenic protein medin is associated with a range of vascular diseases, including aortic aneurysms, vascular dementia, and Alzheimer's disease. Medin accumulates in the vasculature with age, leading to endothelial dysfunction through oxidative and nitrative stress and inducing pro-inflammatory activation. Medin is a cleavage product from the C2 domain of MfgE8. The exact mechanism of medin production from MfgE8 is unknown, with crystal structures of homologous C2 domains suggesting that the cleavage sites are buried, requiring a conformational transition for medin production. Molecular dynamics simulations can explore a wide range of conformations, from small-scale bond rotations to large-scale changes like protein folding or ligand binding. This study employed a combination of full-atom and coarse-grained molecular dynamics simulations, along with CONCOORD- and AlphaFold2-generated models, to investigate MfgE8 conformations and their implications for medin cleavage site accessibility. The simulations revealed that MfgE8 tends to adopt a compact conformation with the RGD motif, important for cell attachment within the N-terminal domain, and the medin region in the C-terminal domain close in proximity. Formation of this compact structure is facilitated by interdomain electrostatic interactions that promote stability and in turn decrease the solvent-accessible surface area of the medin region and particularly the C-terminal medin cleavage site. This data enhances current knowledge on medin generation to propose that alterations in local environmental conditions, possibly through changes in glycosylation or other post-translational modifications are required to induce MfgE8 to unfold partially or fully: this would result in enhanced accessibility of the cleavage sites and therefore enable medin generation.

PER2 binding to PDK1 enhances the cisplatin sensitivity of oral squamous cell carcinoma through inhibition of the AKT/mTOR pathway

Cisplatin (CDDP) is a cornerstone chemotherapeutic agent used to treat oral squamous cell carcinoma (OSCC) and many solid cancers. However, the mechanisms underlying tumor resistance to CDDP obscure the enhancement of its therapeutic efficacy. In this study, we unveil diminished expression of the biological clock gene PER2 in OSCC, negatively correlated with the expression of multidrug resistance protein 1 (MDR1) and multidrug resistance-associated protein 1 (MRP1). The overexpression of PER2 suppressed MDR1 and MRP1 expression and increased intracellular CDDP levels and DNA damage, thereby bolstering OSCC cell sensitivity to CDDP. In vivo tumorigenic assays corroborated that PER2 overexpression notably increased OSCC sensitivity to CDDP, augmenting the suppression of OSCC tumorigenesis. Co-immunoprecipitation, GST pull-down, and cycloheximide tracking assays revealed that PER2, via its C-terminal domain, bound to and diminishes PDK1 stability. The degradation of PDK1 was further dependent on the suppression of the AKT/mTOR pathway to enhance the sensitivity of OSCC cells to CDDP. Our study supports PER2 as a target for improving CDDP sensitivity in OSCC, and the combination of PER2 and CDDP is a novel strategy with potential clinical therapeutic value.

Caprin1 Bridges PRMT1 to G3BP1 and Spaces Them to Ensure Proper Stress Granule Formation

Stress granules (SGs) are dynamic biomolecular condensates that form in the cytoplasm in response to cellular stress, encapsulating proteins and RNAs. Methylation is a key factor in the assembly of SGs, with PRMT1, which acts as an arginine methyltransferase, localizing to SGs. However, the precise mechanism of PRMT1 localization within SGs remains unknown. In this study, we identified that Caprin1 plays a primary role in the recruitment of PRMT1 to SGs, particularly through its C-terminal domain. Our findings demonstrate that Caprin1 serves a dual function as both a linker, facilitating the formation of a PRMT1-G3BP1 complex, and as a spacer, preventing the aberrant formation of SGs under non-stress conditions. This study sheds new lights on the regulatory mechanisms governing SG formation and suggests that Caprin1 plays a critical role in cellular responses to stress.

Structures of BlEst2 from Bacillus licheniformis in its propeptide and mature forms reveal autoinhibitory effects of the C-terminal domain.

Carboxylesterases comprise a major class of α/β-fold hydrolases responsible for the cleavage and formation of ester bonds. Found ubiquitously in nature, these enzymes are crucial for the metabolism of both endogenous and exogenous carboxyl esters in animals, plants and microorganisms. Beyond their essential physiological roles, carboxylesterases stand out as one of the important classes of biocatalysts for biotechnology. BlEst2, an enzyme previously classified as Bacillus licheniformis esterase, remains largely uncharacterized. In the present study, we elucidate the structural biology, molecular dynamics and biochemical features of BlEst2. Our findings reveal a canonical α/β-hydrolase fold similar to the ESTHER block L of lipases, further augmented by two additional accessory C-terminal domains. Notably, the catalytic domain demonstrates two insertions, which occupy conserved locations in α/β-hydrolase proteins and commonly form the lid domain in lipase structures. Intriguingly, our in vitro cleavage of C-terminal domains revealed the structure of the active form of BlEst2. Upon activation, BlEst2 showed a markedly elevated hydrolytic activity. This observation implies that the intramolecular C-terminal domain serves as a regulatory intramolecular inhibitor. Interestingly, despite exhibiting esterase-like activity, BlEst2 structural characteristics align more closely with lipases. This suggests that BlEst2 could potentially represent a previously unrecognized subgroup within the realm of carboxyl ester hydrolases.

Deciphering the enigmatic PilY1 of Acidithiobacillus thiooxidans: An in silico analysis

Thirty years since the first report on the PilY1 protein in bacteria, only the C-terminal domain has been crystallized; there is no study in which the N-terminal domain, let alone the complete protein, has been crystallized. In our laboratory, we are interested in characterizing the Type IV Pili (T4P) of Acidithiobacillus thiooxidans. We performed an in silico characterization of PilY1 and other pilins of the T4P of this acidophilic bacterium. In silico characterization is crucial for understanding how proteins adapt and function under extreme conditions. By analyzing the primary and secondary structures of proteins through computational methods, researchers can gain valuable insights into protein stability, key structural features, and unique amino acid compositions that contribute to resilience in harsh environments. Here, it is presented a description of the particularities of At. thiooxidans PilY1 through predictor software and homology data. Our results suggest that PilY1 from At. thiooxidans may have the same role as has been described for other PilY1 associated with T4P in neutrophilic bacteria; also, its C-terminal interacts (interface interaction) with the minor pilins PilX, PilW and PilV. The N-terminal region comprises domains such as the vWA and the MIDAS, involved in signaling, ligand-binding, and protein-protein interaction. In fact, the vWA domain has intrinsically disordered regions that enable it to maintain its structure over a wide pH range, not only at extreme acidity to which At. thiooxidans is adapted. The results obtained helped us design the correct methodology for its heterologous expression. This allowed us partially experimentally characterize it by obtaining the N-terminal domain recombinantly and evaluating its acid stability through fluorescence spectroscopy. The data suggest that it remains stable across pH changes. This work thus provides guidance for the characterization of extracellular proteins from extremophilic organisms.

Hdm2 disrupts HdmX-mediated nuclear export of p53 by sequestering it in nucleus

Dysfunction of the tumor suppressor p53 occurs in most human cancers, Hdm2 and HdmX play critical roles in p53 inactivation and degradation. Under unstressed conditions, HdmX binds to p53 like Hdm2, but HdmX cannot directly induce p53 degradation. Moreover, HdmX has been reported to stimulate Hdm2-mediated ubiquitination and degradation of p53. Here we reported that HdmX promoted the nuclear export of p53 independent of Hdm2 in living cells using FRET technology. Whereas, Hdm2 impeded HdmX-mediated nuclear export of p53 by sequestering it in nucleus. Interestingly, the C-terminal RING domain mutant Hdm2C464A formed heterooligomers with p53 in nucleus, which was inhibited by HdmX. The heterooligomers were located near PML-NBs. This study indicate that the nuclear Hdm2-HdmX interaction aborts the HdmX-mediated nuclear export of p53.

β-catenin mediates endodermal commitment of human ES cells via distinct transactivation functions

Backgroundβ-catenin, acting as the core effector of canonical Wnt signaling pathway, plays a pivotal role in controlling lineage commitment and the formation of definitive endoderm (DE) during early embryonic development. Despite extensive studies using various animal and cell models, the β-catenin-centered regulatory mechanisms underlying DE formation remain incompletely understood, partly due to the rapid and complex cell fate transitions during early differentiation.ResultsIn this study, we generated new CTNNB1-/- human ES cells (hESCs) using CRISPR-based insertional gene disruption approach and systematically rescued the DE defect in these cells by introducing various truncated or mutant forms of β-catenin. Our analysis showed that a truncated β-catenin lacking both N- and C-terminal domains (ΔN148C) could robustly rescue the DE formation, whereas hyperactive β-catenin mutants with S33Y mutation or N-terminal deletion (ΔN90) had limited ability to induce DE lineage. Notably, the ΔN148C mutant exhibited significant nuclear translocation that was positively correlated with successful DE rescue. Transcriptomic analysis further uncovered that two weak β-catenin mutants lacking the C-terminal transactivation domain (CTD) activated primitive streak (PS) genes, whereas the hyperactive β-catenin mutants activated mesoderm genes.ConclusionOur study uncovered an unconventional regulatory function of β-catenin through weak transactivation, indicating that the levels of β-catenin activity determine the lineage bifurcation from mesendoderm into endoderm and mesoderm.

Novel hypermorphic variants in IRF2BP2 identified in patients with common variable immunodeficiency and autoimmunity

The interferon regulatory factor 2 binding protein 2 (IRF2BP2) is a transcriptional regulator, functioning a transcriptional corepressor by interacting with the interferon regulatory factor-2. The ubiquitous expression of IRF2BP2 by diverse cell types and tissues suggests its potential involvement in different cell signalling pathways. Variants inIRF2BP2have been recently identified to cause familial common variable immunodeficiency (CVID) characterized by immune dysregulation.This study investigated three rare novel variants inIRF2BP2, identified in patients with primary antibody deficiency and autoimmunity by whole exome-sequencing (WES). Following transient overexpression of EGFP-fused mutants in HEK293 cells and transfection in Jurkat cell lines, we used fluorescence microscopy, real-time PCR and Western blotting to analyze their effects on IRF2BP2 expression, subcellular localization, nuclear translocation of IRF2, and the transcriptional activation of NFκB1(p50).We found altered IRF2BP2 mRNA and protein expression levels in the mutants compared to the wild type after IRF2BP2 overexpression. In confocal fluorescence microscopy, variants in the C-terminal RING finger domain showed an irregular aggregate formation and distribution instead of the expected nuclear localization compared to the variants in the N-terminal zinc finger domain and their wildtype counterpart. Immunoblotting revealed an impaired IRF2 and NFκB1 (p50) nuclear localization in the mutants compared to the IRF2BP2 wildtype counterpart. LPS stimulation reduced IRF2BP2 mRNA expression in the variants compared to the wild type.Our findings significantly contribute to understanding the clinical significance of IRF2BP2 mutations in the pathogenesis of immunodeficiency and immune dysregulation. We observed impairment of the nuclear translocation of IRF2 and NFκB1 (p50) due to the upregulation of IRF2BP2, potentially affecting specific gene expressions involved in immune regulation.

BCL-2 and BOK regulate apoptosis by interaction of their C-terminal transmembrane domains

The Bcl-2 family controls apoptosis by direct interactions of pro- and anti-apoptotic proteins. The principle mechanism is binding of the BH3 domain of pro-apoptotic proteins to the hydrophobic groove of anti-apoptotic siblings, which is therapeutically exploited by approved BH3-mimetic anti-cancer drugs. Evidence suggests that also the transmembrane domain (TMD) of Bcl-2 proteins can mediate Bcl-2 interactions. We developed a highly-specific split luciferase assay enabling the analysis of TMD interactions of pore-forming apoptosis effectors BAX, BAK, and BOK with anti-apoptotic Bcl-2 proteins in living cells. We confirm homotypic interaction of the BAX-TMD, but also newly identify interaction of the TMD of anti-apoptotic BCL-2 with the TMD of BOK, a peculiar pro-apoptotic Bcl-2 protein. BOK-TMD and BCL-2-TMD interact at the endoplasmic reticulum. Molecular dynamics simulations confirm dynamic BOK-TMD and BCL-2-TMD dimers and stable heterotetramers. Mutation of BCL-2-TMD at predicted key residues abolishes interaction with BOK-TMD. Also, inhibition of BOK-induced apoptosis by BCL-2 depends specifically on their TMDs. Thus, TMDs of Bcl-2 proteins are a relevant interaction interface for apoptosis regulation and provide a novel potential drug target.

Novel transcriptional regulation of the GAP promoter in Pichia pastoris towards high expression of heterologous proteins

BackgroundPichia pastoris (Komagataella phaffii) is a promising production host, but the usage of methanol limits its application in the medicine and food industries.ResultsTo improve the constitutive expression of heterologous proteins in P. pastoris, four new potential transcription regulators (Loc1p, Msn2p, Gsm1p, Hot1p) of the glyceraldehyde triphosphate dehydrogenase promoter (pGAP) were revealed in this study by using cellulase E4 as reporter gene. On this basis, a series of P. pastoris strains with knockout or overexpression of transcription factors were constructed and the deletion of transcription factor binding sites on pGAP was confirmed. The results showed that Loc1p and Msn2p can inhibit the activity of pGAP, while Gsm1p and Hot1p can enhance the activity of pGAP; Loc1p, Gsm1p and Hot1p can bind directly to pGAP, while Msn2p must be treated to expose the C-terminal domain to bind to pGAP. Moreover, manipulating a single transcription factor led to a 0.96-fold to 2.43-fold increase in xylanase expression. In another model protein, aflatoxin oxidase, knocking out Loc1 based on AFO-∆Msn2 strain resulted in a 0.63-fold to 1.4-fold increase in expression. It can be demonstrated that the combined use of transcription factors can further improve the expression of exogenous proteins in P. pastoris.ConclusionThese findings will contribute to the construction of pGAP-based P. pastoris systems towards high expression of heterologous proteins, hence improving the application potential of yeast.

FhlA is a Formate Binding Protein.

Escherichia coli uses glycolysis and mixed acid fermentation and produces formate as by product. One system E. coli uses for formate oxidation is formate hydrogen lyase complex (FHL). The expression of the FHL complex is dependent on formate and regulated by the transcriptional regulator FhlA. The structure of FhlA is composed of three domains. The N-terminal domain is putatively responsible for formate binding and FhlA oligomerization as a tetramer, the central portion of FhlA contains a AAA+ domain that hydrolyzes ATP, and the C-terminal domain binds DNA. Formate enhances FhlA-mediated expression of FHL; however, FhlA direct interaction with formate has never been demonstrated. Formate-protein interactions are challenging to assess, due to the small and ubiquitous nature of the molecule. Here, we have developed three techniques to assess formate-protein interaction. We use these techniques to confirm that FhlA binds formate in the N-terminal domain in vitro, and that this interaction is partially dependent on residues E183 and E363, consistent with previous reports. This study is a proof of concept that these techniques can be used to assess other formate-protein interactions.

Transient Structural Properties of the Rho GDP-Dissociation Inhibitor.

Rho GTPases, master spatial regulators of a wide range of cellular processes, are orchestrated by complex formation with guanine nucleotide dissociation inhibitors (RhoGDIs). These have been thought to possess an unstructured N-terminus that inhibits nucleotide exchange of their client upon binding/folding. Via NMR analyses, molecular dynamics simulations, and biochemical assays, we reveal instead pertinent structural properties transiently maintained both, in the presence and absence of the client, imposed onto the terminus context-specifically by modulating interactions with the surface of the folded C-terminal domain. These observations revise the long-standing textbook picture of the GTPases' mechanism of membrane extraction. Rather than by a disorder-to-order transition upon binding of an inhibitory peptide, the intricate and highly selective extraction process of RhoGTPases is orchestrated via a dynamic ensemble bearing preformed transient structural properties, suitably modulated by the specific surrounding along the multi-step process.

Elf1 promotes transcription-coupled repair in yeast by using its C-terminal domain to bind TFIIH

Transcription coupled-nucleotide excision repair (TC-NER) removes DNA lesions that block RNA polymerase II (Pol II) transcription. A key step in TC-NER is the recruitment of the TFIIH complex, which initiates DNA unwinding and damage verification; however, the mechanism by which TFIIH is recruited during TC-NER, particularly in yeast, remains unclear. Here, we show that the C-terminal domain (CTD) of elongation factor-1 (Elf1) plays a critical role in TC-NER in yeast by binding TFIIH. Analysis of genome-wide repair of UV-induced cyclobutane pyrimidine dimers (CPDs) using CPD-seq indicates that the Elf1 CTD in yeast is required for efficient TC-NER. We show that the Elf1 CTD binds to the pleckstrin homology (PH) domain of the p62 subunit of TFIIH in vitro, and identify a putative TFIIH-interaction region (TIR) in the Elf1 CTD that is important for PH binding and TC-NER. The Elf1 TIR shows functional, structural, and sequence similarities to a conserved TIR in the mammalian UV sensitivity syndrome A (UVSSA) protein, which recruits TFIIH during TC-NER in mammalian cells. These findings suggest that the Elf1 CTD acts as a functional counterpart to mammalian UVSSA in TC-NER by recruiting TFIIH in response to Pol II stalling at DNA lesions.

New Class of Hsp90 C-Terminal Domain Inhibitors with Anti-tumor Properties against Triple-Negative Breast Cancer.

Triple-negative breast cancer (TNBC) remains a treatment challenge and requires innovative therapies. Hsp90, crucial for the stability of numerous oncogenic proteins, has emerged as a promising therapeutic target. In this study, we present the optimization of the Hsp90 C-terminal domain (CTD) inhibitor TVS21. Biochemical methods, NMR binding studies, and molecular modeling were employed to investigate the binding of representative analogs to Hsp90. The newly synthesized analogs showed increased antiproliferative activity in breast cancer cell lines, including the MDA-MB-231 TNBC cell line. Compounds 89 and 104 proved to be the most effective, inducing apoptosis, slowing proliferation, and degrading key oncogenic proteins without inducing a heat shock response. In vivo, compound 89 showed comparable efficacy to the clinical candidate AUY922 and a better safety profile in a TNBC xenograft model. These results highlight the promise of Hsp90 CTD inhibitors for TNBC therapy, potentially filling a significant treatment gap.

Oxidative stress elicits the remodeling of vimentin filaments into biomolecular condensates

The intermediate filament protein vimentin performs an essential role in cytoskeletal interplay and dynamics, mechanosensing and cellular stress responses. In pathology, vimentin is a key player in tumorigenesis, fibrosis and infection. Vimentin filaments undergo distinct and versatile reorganizations, and behave as redox sensors. The vimentin monomer possesses a central α-helical rod domain flanked by N- and C-terminal low complexity domains. Interactions between this type of domains play an important function in the formation of phase-separated biomolecular condensates, which in turn are critical for the organization of cellular components. Here we show that several oxidants, including hydrogen peroxide and diamide, elicit the remodeling of vimentin filaments into small particles. Oxidative stress elicited by diamide induces a fast dissociation of filaments into circular, motile dots, which requires the presence of the single vimentin cysteine residue, C328. This effect is reversible, and filament reassembly can occur within minutes of oxidant removal. Diamide-elicited vimentin droplets recover fluorescence after photobleaching. Moreover, fusion of cells expressing differentially tagged vimentin allows the detection of dots positive for both tags, indicating that vimentin dots merge upon cell fusion. The aliphatic alcohol 1,6-hexanediol, known to alter interactions between low complexity domains, readily dissolves diamide-elicited vimentin dots at low concentrations, in a C328 dependent manner, and hampers reassembly. Taken together, these results indicate that vimentin oxidation promotes a fast and reversible filament remodeling into biomolecular condensate-like structures, and provide primary evidence of its regulated phase separation. Moreover, we hypothesize that filament to droplet transition could play a protective role against irreversible damage of the vimentin network by oxidative stress.

An MDM2 degrader shows potent cytotoxicity to MDM2-overexpressing acute lymphoblastic leukemia cells with minimal toxicity to normal cells/tissues

The MDM2 oncogene is amplified and/or overexpressed in various human cancers and elevated expression of MDM2 protein acts as a survival factor promoting cancer progression through both p53-dependent and -independent pathways. Here, we report a novel small-molecule chemical compound (MX69-102) that we identified to induce MDM2 protein degradation, resulting in reactivation of p53, inhibition of XIAP, and potent cell growth inhibition and apoptosis in MDM2-overexpressing acute lymphoblastic leukemia (ALL) in vitro and in vivo. We have previously identified a compound (MX69) that binds to the MDM2 C-terminal RING domain and induces MDM2 protein degradation. In the present study, we performed structural modifications of MX69 and selected analog MX69-102, showing increased MDM2-targeting activity. MX69-102 exhibited significantly enhanced inhibitory and apoptotic effects on a group of MDM2-overexpressing ALL cell lines in vitro with IC50 values of about 0.2 μM, representing an approximately 38-fold increase in activity compared to MX69. MX69-102 also showed effective inhibition on xenografted human MDM2-overexpressing ALL in SCID mice. Importantly, MX69-102 had minimal or no inhibitory effect on normal human hematopoiesis in vitro and was very well tolerated in vivo in animal models. Based on the strong inhibitory and apoptotic activity against MDM2-overexpressing ALL, along with minimal or no toxicity to normal cells/tissues, MX69-102 is a candidate for further development as a novel MDM2-targeted therapeutic drug for refractory/MDM2-overexpressing ALL.

MUT-7 exoribonuclease activity and localization are mediated by an ancient domain.

The MUT-7 family of 3'-5' exoribonucleases is evolutionarily conserved across the animal kingdom and plays essential roles in small RNA production in the germline. Most MUT-7 homologues carry a C-terminal domain of unknown function named MUT7-C appended to the exoribonuclease domain. Our analysis shows that the MUT7-C is evolutionary ancient, as a minimal version of the domain exists as an individual protein in prokaryotes. In animals, MUT7-C has acquired an insertion that diverged during evolution, expanding its functions. Caenorhabditis elegans MUT-7 contains a specific insertion within MUT7-C, which allows binding to MUT-8 and, consequently, MUT-7 recruitment to germ granules. In addition, in C. elegans and human MUT-7, the MUT7-C domain contributes to RNA binding and is thereby crucial for ribonuclease activity. This RNA-binding function most likely represents the ancestral function of the MUT7-C domain. Overall, this study sheds light on MUT7-C and assigns two functions to this previously uncharacterized domain.

PP2C phosphatase Pic14 negatively regulates tomato Pto/Prf-triggered immunity by inhibiting MAPK activation.

Type 2C protein phosphatases (PP2Cs) are emerging as important regulators of plant immune responses, although little is known about how they might impact nucleotide-binding, leucine-rich repeat (NLR)-triggered immunity (NTI). We discovered that expression of the PP2C immunity-associated candidate 14 gene (Pic14) is induced upon activation of the Pto/Prf-mediated NTI response in tomato. Pto/Prf recognizes the effector AvrPto translocated into plant cells by the pathogen Pseudomonas syringae pv. tomato (Pst) and activate a MAPK cascade and other responses which together confer resistance to bacterial speck disease. Pic14 encodes a PP2C with an N-terminal kinase-interacting motif (KIM) and a C-terminal phosphatase domain. Upon inoculation with Pst-AvrPto, Pto/Prf-expressing tomato plants with loss-of-function mutations in Pic14 developed less speck disease, specifically in older leaves, compared to wild-type plants. Transient expression of Pic14 in leaves of Nicotiana benthamiana and tomato inhibited cell death typically induced by Pto/Prf and the MAPK cascade members M3Kα and Mkk2. The cell death-suppressing activity of Pic14 was dependent on the KIM and the catalytic phosphatase domain. Pic14 inhibited M3Kα- and Mkk2-mediated activation of immunity-associated MAPKs and Pic14 was shown to be an active phosphatase that physically interacts with and dephosphorylates Mkk2 in a KIM-dependent manner. Together, our results reveal Pic14 as an important negative regulator of Pto/Prf-triggered immunity by interacting with and dephosphorylating Mkk2.