Explore the vast range of titles available.

PPM1D encodes a serine/threonine phosphatase that regulates numerous pathways including the DNA damage response and p53. Activating mutations and amplification of PPM1D are found across numerous cancer types. GSK2830371 is a potent and selective allosteric inhibitor of PPM1D, but its mechanism of binding and inhibition of catalytic activity are unknown. Here we use computational, biochemical and functional genetic studies to elucidate the molecular basis of GSK2830371 activity. These data confirm that GSK2830371 binds an allosteric site of PPM1D with high affinity. By further incorporating data from hydrogen deuterium exchange mass spectrometry and sedimentation velocity analytical ultracentrifugation, we demonstrate that PPM1D exists in an equilibrium between two conformations that are defined by the movement of the flap domain, which is required for substrate recognition. A hinge region was identified that is critical for switching between the two conformations and was directly implicated in the high-affinity binding of GSK2830371 to PPM1D. We propose that the two conformations represent active and inactive forms of the protein reflected by the position of the flap, and that binding of GSK2830371 shifts the equilibrium to the inactive form. Finally, we found that C-terminal truncating mutations proximal to residue 400 result in destabilization of the protein via loss of a stabilizing N- and C-terminal interaction, consistent with the observation from human genetic data that nearly all PPM1D mutations in cancer are truncating and occur distal to residue 400. Taken together, our findings elucidate the mechanism by which binding of a small molecule to an allosteric site of PPM1D inhibits its activity and provides insights into the biology of PPM1D. In this work, the authors report a sophisticated combination of genetic, biophysical, and biochemical analyses to identifies the cycling conformational states of PPM1D. The findings reveal how an allosteric inhibitor locks the protein into a conformationally inactive state, and explain the distribution of PPM1D activating mutations in cancer.

Serine/threonine kinases (STKs) and serine/threonine phosphatases (STPs) are widely present across various organisms and play crucial roles in regulating cellular processes such as growth, proliferation, signal transduction, and other physiological functions. Recent research has increasingly focused on the regulation of STKs and STPs in bacteria. STKs have been well studied, identified and characterized in a variety of bacterial species. However, the role of STPs in bacteria remains less understood, and the number of proteins characterized is limited. It has been found that most of the STPs characterized in bacteria were Mg 2+ /Mn 2+ dependent 2C protein phosphatases (PP2Cs), but the evolutionary relationship and taxonomic distribution of bacterial PP2C phosphatases were still not fully elucidated. In this study, we utilized bacterial PP2C phosphatase sequences from the InterPro database to perform a phylogenetic analysis, categorizing the family into five groups. Based on this classification, we examined the evolutionary relationships, species distribution, sequence and structural variations, and domain distribution characteristics of bacterial PP2C phosphatases. Our analysis uncovered evidence of a common evolutionary origin for bacterial PP2C phosphatases. These findings advance the understanding of PP2C phosphatases, offering valuable insights for future functional studies of bacterial serine/threonine phosphatases and aiding in the design of targeted therapeutics for pathogenic bacteria.

DNA damage response (DDR) pathway protects cells from genome instability and prevents cancer development. Tumor suppressor p53 is a key molecule that interconnects DDR, cell cycle checkpoints, and cell fate decisions in the presence of genotoxic stress. Inactivating mutations in TP53 and other genes implicated in DDR potentiate cancer development and also influence the sensitivity of cancer cells to treatment. Protein phosphatase 2C delta (referred to as WIP1) is a negative regulator of DDR and has been proposed as potential pharmaceutical target. Until recently, exploitation of WIP1 inhibition for suppression of cancer cell growth was compromised by the lack of selective small-molecule inhibitors effective at cellular and organismal levels. Here, we review recent advances in development of WIP1 inhibitors and discuss their potential use in cancer treatment.

Clade A protein phosphatase 2Cs (PP2C-As) play crucial roles in plant stress responses. Although the ABA receptors PYLs inhibit PP2C-As in an ABA-dependent manner, other modulators of these phosphatases remain largely unknown. Here, we identify the FORKED-LIKE 7 (FL7) protein as a broad PP2C-A interactor that effectively suppresses PP2C activity through an ABA-independent, noncompetitive mechanism. By inhibiting PP2C-A activity, FL7 positively regulates osmotic tolerance and plant immunity in an ABA-independent manner. The N-terminal auxin canalisation (AC) domain of FL7 is required for its PP2C-As inhibitory activity. Further evolutionary analyses reveal that FL7 homologues containing an AC domain belong to an ancient family that emerged in a common ancestor of Klebsormidiophyceae algae and land plants. Genetic analyses indicate that algal FL7 homologues have a conserved function as PP2C-A inhibitors. Our study reveals an ABA-independent layer of PP2C-A modulation that regulates biotic and abiotic stress responses, which is likely conserved across a billion years of streptophyte evolution and predated the PYL-ABA regulation established in the common ancestor of land plants. Li et al. reveal an ABA-independent layer of PP2C-A modulation that regulates both biotic and abiotic stress responses. This mechanism is conserved across approximately a billion years and predates the PYL-ABA regulatory system established in the common ancestor of land plants.

The plant protein phosphatase 2C (PP2C) plays an irreplaceable role in phytohormone signaling, developmental processes, and manifold stresses. However, information about the PP2C gene family in tomato (Solanum lycopersicum) is relatively restricted. In this study, a genome-wide investigation of the SlPP2C gene family was performed. A total of 92 SlPP2C genes were identified, they were distributed on 11 chromosomes, and all the SlPP2C proteins have the type 2C phosphatase domains. Based on phylogenetic analysis of PP2C genes in Arabidopsis, rice, and tomato, SlPP2C genes were divided into eight groups, designated A–H, which is also supported by the analyses of gene structures and protein motifs. Gene duplication analysis revealed that the duplication of whole genome and chromosome segments was the main cause of SLPP2Cs expansion. A total of 26 cis-elements related to stress, hormones, and development were identified in the 3 kb upstream region of these SlPP2C genes. Expression profile analysis revealed that the SlPP2C genes display diverse expression patterns in various tomato tissues. Furthermore, we investigated the expression patterns of SlPP2C genes in response to Ralstonia solanacearum infection. RNA-seq and qRT-PCR data reveal that nine SlPP2Cs are correlated with R. solanacearum. The above evidence hinted that SlPP2C genes play multiple roles in tomato and may contribute to tomato resistance to bacterial wilt. This study obtained here will give an impetus to the understanding of the potential function of SlPP2Cs and lay a solid foundation for tomato breeding and transgenic resistance to plant pathogens.

Protein phosphatases (PPs) are a class of enzymes that play a critical role in cellular regulation by catalyzing the removal of phosphate groups from proteins. This dephosphorylation process is essential for controlling and modulating various cellular functions, including signal transduction, cell cycle progression, metabolic regulation, and stress responses. This study focuses on the comprehensive genomic identification, evolutionary analysis, and transcript profiling of the PP2C gene family within Solanum lycopersicum , an economically significant crop with substantial agricultural and nutritional importance. A total of 95 PP2C members have been identified in tomato, which was divided into 12 subgroups. An evolutionary comparison of the tomato PP2C members with other plant species demonstrated that they shared a common ancestor. A total of 14 SlPP2C s have arisen from segmental duplication events, while no tandem duplication was detected. Certain SlPP2C genes exhibited unique expression patterns in specific tissues, with only a limited number of SlPP2C genes being expressed in all tissues, while almost all SlPP2Cs are upregulated during the flowering stage. Gene expression analysis revealed elevated transcript levels of SlPP2C22 , SlPP2C30 , and SlPP2C52 during drought stress. An increase in total PP2C enzyme activity was also observed which indicates their significance in drought stress. These findings add to the comprehension of the evolutionary history and significance of tomato PP2C in managing abiotic stress and pave the way for additional verification of the functional aspect of these PP2C genes in tomato.

We conducted a two-sample Mendelian randomization study to test the hypothesis that raised plasma levels of the branched-chain amino acids isoleucine, leucine, and valine are associated with Alzheimer’s disease (AD). From a genome-wide association study of 16,596 individuals of European ancestry, we obtained summary statistics for four independent single nucleotide polymorphisms (SNPs) associated with isoleucine levels and one SNP associated with both leucine and valine levels at genome-wide significance. Summary statistics of the associations of the five SNPs with AD were obtained from the International Genomics of Alzheimer’s Project (17,008 AD cases and 37,154 controls). Based on four SNPs, the odds ratio of AD per genetically predicted one standard deviation higher isoleucine levels was 1.35 (95% CI, 1.08–1.69; p  = 0.007). The leucine- and valine-raising allele was not associated with AD ( p  = 0.46). These data suggest that a genetic predisposition to raised plasma isoleucine levels is positively associated with AD.

Inducible transcriptional programs mediate the regulation of key biological processes and organismal functions. Despite their complexity, cells have evolved mechanisms to precisely control gene programs in response to environmental cues to regulate cell fate and maintain normal homeostasis. Upon stimulation with proinflammatory cytokines such as tumor necrosis factor-α (TNF), the master transcriptional regulator nuclear factor (NF)-κB utilizes the PPM1G/PP2Cγ phosphatase as a coactivator to normally induce inflammatory and cell survival programs. However, how PPM1G activity is precisely regulated to control NF-κB transcription magnitude and kinetics remains unknown. Here, we describe a mechanism by which the ARF tumor suppressor binds PPM1G to negatively regulate its coactivator function in the NF-κB circuit thereby promoting insult resolution. ARF becomes stabilized upon binding to PPM1G and forms a ternary protein complex with PPM1G and NF-κB at target gene promoters in a stimuli-dependent manner to provide tunable control of the NF-κB transcriptional program. Consistently, loss of ARF in colon epithelial cells leads to up-regulation of NF-κB antiapoptotic genes upon TNF stimulation and renders cells partially resistant to TNF-induced apoptosis in the presence of agents blocking the antiapoptotic program. Notably, patient tumor data analysis validates these findings by revealing that loss of ARF strongly correlates with sustained expression of inflammatory and cell survival programs. Collectively, we propose that PPM1G emerges as a therapeutic target in a variety of cancers arising from ARF epigenetic silencing, to loss of ARF function, as well as tumors bearing oncogenic NF-κB activation.

Background PPM1D (protein phosphatase Mg 2 ⁺/Mn 2 ⁺ dependent 1D) is a Ser/Thr phosphatase that negatively regulates p53 and functions as an oncogenic driver. Its gene amplification and overexpression are frequently observed in various malignancies and disruption of PPM1D degradation has also been reported as a cause of cancer progression. However, the precise mechanisms regulating PPM1D stability remain to be elucidated. Methods PPM1D stability and degradation pathways were examined using cycloheximide chase assays in multiple cell lines. Proteasome and lysosome inhibitors were used to determine the degradation mechanism, while ubiquitination dependency was assessed using TAK-243, an E1 ubiquitin-activating enzyme inhibitor. In vitro degradation assays with purified 20S proteasome were performed to evaluate direct proteasomal degradation. Immunoprecipitation followed by mass spectrometry was performed to identify proteasomal regulators of PPM1D, with their functional roles validated through knockdown experiments. Finally, cell viability assays were conducted to assess the therapeutic potential of combined proteasome and PPM1D inhibition. Results Cycloheximide chase assays demonstrated that wild-type PPM1D is a short-lived protein, whereas a C-terminal truncation mutant exhibits increased stability. PPM1D undergoes rapid, ubiquitin-independent proteasomal degradation via its C-terminal 35 amino acid residues. Additionally, the region spanning residues 450–501 is necessary for ubiquitination-mediated suppression of the ubiquitin-independent degradation pathway. We also found that PPM1D is directly degraded by the 20S proteasome, with the regulatory proteasome subunits PSMD14 and PSME3 acting as activators in this process. Proteasome inhibition resulted in PPM1D accumulation, potentially reducing therapeutic efficacy. Combined proteasome and PPM1D inhibition synergistically enhanced the antitumor effect. Conclusions The rapid degradation of the cancer driver PPM1D is achieved through direct recognition by the proteasome, and proteasome inhibitors may reduce therapeutic efficacy due to the accumulation of PPM1D. PPM1D may serve as a suitable model substrate for elucidating the mechanism of ubiquitin-independent proteasomal degradation and represents a potential novel therapeutic target for cancer treatment based on proteasome inhibition.

Dramatic cellular reorganization in mitosis critically depends on the timely and temporal phosphorylation of a broad range of proteins, which is mediated by the activation of the mitotic kinases and repression of counteracting phosphatases. The mitosis-to-interphase transition, which is termed mitotic exit, involves the removal of mitotic phosphorylation by protein phosphatases. Although protein phosphatase 1 (PP1) and protein phosphatase 2A (PP2A) drive this reversal in animal cells, the phosphatase network associated with ordered bulk dephosphorylation in mitotic exit is not fully understood. Here, we describe a new mitotic phosphatase relay in which Wip1/PPM1D phosphatase activity is essential for chromosomal passenger complex (CPC) translocation to the anaphase central spindle after release from the chromosome via PP1-mediated dephosphorylation of histone H3T3. Depletion of endogenous Wip1 and overexpression of the phosphatase-dead mutant disturbed CPC translocation to the central spindle, leading to failure of cytokinesis. While Wip1 was degraded in early mitosis, its levels recovered in anaphase and the protein functioned as a Cdk1-counteracting phosphatase at the anaphase central spindle and midbody. Mechanistically, Wip1 dephosphorylated Thr-59 in inner centromere protein (INCENP), which, subsequently bound to MKLP2 and recruited other components to the central spindle. Furthermore, Wip1 overexpression is associated with the overall survival rate of patients with breast cancer, suggesting that Wip1 not only functions as a weak oncogene in the DNA damage network but also as a tumor suppressor in mitotic exit. Altogether, our findings reveal that sequential dephosphorylation of mitotic phosphatases provides spatiotemporal regulation of mitotic exit to prevent tumor initiation and progression.