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Proceedings of the National Academy of... Apr 2024Dephosphorylation of pSer51 of the α subunit of translation initiation factor 2 (eIF2α) terminates signaling in the integrated stress response (ISR). A trimeric...
Dephosphorylation of pSer51 of the α subunit of translation initiation factor 2 (eIF2α) terminates signaling in the integrated stress response (ISR). A trimeric mammalian holophosphatase comprised of a protein phosphatase 1 (PP1) catalytic subunit, the conserved C-terminally located ~70 amino acid core of a substrate-specific regulatory subunit (PPP1R15A/GADD34 or PPP1R15B/CReP) and G-actin (an essential cofactor) efficiently dephosphorylate eIF2α in vitro. Unlike their viral or invertebrate counterparts, with whom they share the conserved 70 residue core, the mammalian PPP1R15s are large proteins of more than 600 residues. Genetic and cellular observations point to a functional role for regions outside the conserved core of mammalian PPP1R15A in dephosphorylating its natural substrate, the eIF2 trimer. We have combined deep learning technology, all-atom molecular dynamics simulations, X-ray crystallography, and biochemistry to uncover binding of the γ subunit of eIF2 to a short helical peptide repeated four times in the functionally important N terminus of human PPP1R15A that extends past its conserved core. Binding entails insertion of Phe and Trp residues that project from one face of an α-helix formed by the conserved repeats of PPP1R15A into a hydrophobic groove exposed on the surface of eIF2γ in the eIF2 trimer. Replacing these conserved Phe and Trp residues with Ala compromises PPP1R15A function in cells and in vitro. These findings suggest mechanisms by which contacts between a distant subunit of eIF2 and elements of PPP1R15A distant to the holophosphatase active site contribute to dephosphorylation of eIF2α by the core PPP1R15 holophosphatase and to efficient termination of the ISR in mammals.
Topics: Animals; Humans; Actins; Eukaryotic Initiation Factor-2; Phosphorylation; Protein Phosphatase 1; Protein Processing, Post-Translational
PubMed: 38547060
DOI: 10.1073/pnas.2320013121 -
Frontiers in Plant Science 2024Plant phytochromes, renowned phosphoproteins, are red and far-red photoreceptors that regulate growth and development in response to light signals. Studies on... (Review)
Review
Plant phytochromes, renowned phosphoproteins, are red and far-red photoreceptors that regulate growth and development in response to light signals. Studies on phytochrome phosphorylation postulate that the N-terminal extension (NTE) and hinge region between N- and C-domains are sites of phosphorylation. Further studies have demonstrated that phosphorylation in the hinge region is important for regulating protein-protein interactions with downstream signaling partners, and phosphorylation in the NTE partakes in controlling phytochrome activity for signal attenuation and nuclear import. Moreover, phytochrome-associated protein phosphatases have been reported, indicating a role of reversible phosphorylation in phytochrome regulation. Furthermore, phytochromes exhibit serine/threonine kinase activity with autophosphorylation, and studies on phytochrome mutants with impaired or increased kinase activity corroborate that they are functional protein kinases in plants. In addition to the autophosphorylation, phytochromes negatively regulate PHYTOCHROME-INTERACTING FACTORs (PIFs) in a light-dependent manner by phosphorylating them as kinase substrates. Very recently, a few protein kinases have also been reported to phosphorylate phytochromes, suggesting new views on the regulation of phytochrome via phosphorylation. Using these recent advances, this review details phytochrome regulation through phosphorylation and highlights their significance as protein kinases in plant light signaling.
PubMed: 38545394
DOI: 10.3389/fpls.2024.1259720 -
Vaccines Feb 2024The gram-negative facultative intracellular pathogen serotype Choleraesuis, also known as . Choleraesuis, is a major financial loss for the pig business. C500 is a...
The gram-negative facultative intracellular pathogen serotype Choleraesuis, also known as . Choleraesuis, is a major financial loss for the pig business. C500 is a vaccine strain that has been used for preventing . Choleraesuis infection in pigs for many years in China. Although it possessed good immunogenicity and protection efficacy, it still showed severe side effects. The truncation of the key gene in C500 was believed to take the major responsibility for its attenuation. To achieve a good balance between attenuation and immunogenicity, was restored to an active state, and other essential virulent genes of , , , and were evaluated for their effects of deletion on safety and immunogenicity. Animal experiments demonstrated that C5001 (C500 Δ) and C5002 (C500 Δ) showed an excellent ability to induce an immune response. To further decrease the endotoxic activity, the combination mutations of Δ Δ::P Δ were introduced into the mutant strains to generate 1'-dephosphorylated lipid A. Animal experiments showed that SC3 (C500 Δ Δ Δ:: P Δ) induced higher levels of IgG and secreted IgA antibodies and provided a higher protection rate than SC1 (C500 Δ Δ:: P Δ) and SC2 (C500 Δ Δ Δ:: P Δ). We also evaluated the ability of SC3 (C500 r Δ Δ Δ:: P Δ) as a vaccine carrier to deliver heterologous protein antigens and polysaccharide antigens. The results indicated that SC3 (C500 r Δ Δ Δ:: P Δ) showed an excellent ability to deliver heterologous antigens and induce the host to produce high levels of antibodies. Together, these results indicate that we constructed a safe and efficient attenuated strain of the . Choleraesuis vaccine, which demonstrated strong resistance to infection with wild-type . Choleraesuis and can be employed as a universal vector for the delivery of recombinant antigens.
PubMed: 38543883
DOI: 10.3390/vaccines12030249 -
Microorganisms Mar 2024A highly active alkaline phosphatase (ALP) of the protein structural family PhoA, from a mussel gut-associated strain of the marine bacterium KMM 296 (CmAP), was found...
A highly active alkaline phosphatase (ALP) of the protein structural family PhoA, from a mussel gut-associated strain of the marine bacterium KMM 296 (CmAP), was found to effectively dephosphorylate lipopolysaccharides (LPS). Therefore, the aim of this work was to perform a comprehensive bioinformatics analysis of the structure, and to suggest the physiological role of this enzyme in marine bacteria of the genus . A scrutiny of the CmAP-like sequences in 36 available genomes revealed nine homologues intrinsic to the subspecies , whereas PhoA of a distant relative JO1 carried an inactive mutation. However, phylogenetic analysis of all available ALP sequences showed that each strain of the genus possesses several ALP variants, mostly the genes encoding for PhoD and PhoX families. The strains have a complete set of four ALP families' genes, namely: PhoA, PafA, PhoX, and two PhoD structures. The species is distinguished by the presence of only three PhoX and PhoD genes. The PhoA proteins are clustered together with the human and squid LPS-detoxifying enzymes. In addition, the predicted PhoA biosynthesis gene cluster suggests its involvement in the control of cellular redox balance, homeostasis, and cell cycle. Apparently, the variety of ALPs in spp. indicates significant adaptability to phosphorus-replete and depleted environments and a notable organophosphate destructor in eco-niches from which they once emerged, including spp. The ALP clusterization and degree of similarity of the genus-specific biosynthetic genes encoding for ectoine and polyketide cluster T1PKS, responsible for sulfated extracellular polysaccharide synthesis, coincide with a new whole genome-based taxonomic classification of the genus . The strains and their ALPs are suggested to be adaptable for use in agriculture, biotechnology and biomedicine.
PubMed: 38543682
DOI: 10.3390/microorganisms12030631 -
Biomolecules Mar 2024Protein phosphatases are primarily responsible for dephosphorylation modification within signal transduction pathways. Phosphatase of regenerating liver-3 (PRL-3) is a... (Review)
Review
Protein phosphatases are primarily responsible for dephosphorylation modification within signal transduction pathways. Phosphatase of regenerating liver-3 (PRL-3) is a dual-specific phosphatase implicated in cancer pathogenesis. Understanding PRL-3's intricate functions and developing targeted therapies is crucial for advancing cancer treatment. This review highlights its regulatory mechanisms, expression patterns, and multifaceted roles in cancer progression. PRL-3's involvement in proliferation, migration, invasion, metastasis, angiogenesis, and drug resistance is discussed. Regulatory mechanisms encompass transcriptional control, alternative splicing, and post-translational modifications. PRL-3 exhibits selective expressions in specific cancer types, making it a potential target for therapy. Despite advances in small molecule inhibitors, further research is needed for clinical application. PRL-3-zumab, a humanized antibody, shows promise in preclinical studies and clinical trials. Our review summarizes the current understanding of the cancer-related cellular function of PRL-3, its prognostic value, and the research progress of therapeutic inhibitors.
Topics: Humans; Signal Transduction; Neoplasms; Protein Tyrosine Phosphatases; Protein Processing, Post-Translational; Phosphoprotein Phosphatases; Cell Line, Tumor
PubMed: 38540761
DOI: 10.3390/biom14030342 -
Science Advances Mar 2024We recently developed a heterobifunctional approach [phosphorylation targeting chimeras (PhosTACs)] to achieve the targeted protein dephosphorylation (TPDephos). Here,...
We recently developed a heterobifunctional approach [phosphorylation targeting chimeras (PhosTACs)] to achieve the targeted protein dephosphorylation (TPDephos). Here, we envisioned combining the inhibitory effects of receptor tyrosine kinase inhibitors (RTKIs) and the active dephosphorylation by phosphatases to achieve dual inhibition of kinases. We report an example of tyrosine phosphatase-based TPDephos and the effective epidermal growth factor receptor (EGFR) tyrosine dephosphorylation. We also used phosphoproteomic approaches to study the signaling transductions affected by PhosTAC-related molecules at the proteome-wide level. This work demonstrated the differential signaling pathways inhibited by PhosTAC compared with the TKI, gefitinib. Moreover, a covalent PhosTAC selective for mutated EGFR was developed and showed its inhibitory potential for dysregulated EGFR. Last, EGFR PhosTACs, consistent with EGFR dephosphorylation profiles, induced apoptosis and inhibited cancer cell viability during prolonged PhosTAC treatment. PhosTACs showcased their potential of modulating RTKs activity, expanding the scope of bifunctional molecule utility.
Topics: Apoptosis; Cell Line, Tumor; ErbB Receptors; Phosphorylation; Signal Transduction; Tyrosine; Tyrosine Kinase Inhibitors; Humans; Proteolysis Targeting Chimera
PubMed: 38536914
DOI: 10.1126/sciadv.adj7251 -
The Journal of Biological Chemistry Apr 2024FOXO1 is a transcription factor and potential tumor suppressor that is negatively regulated downstream of PI3K-PKB/AKT signaling. Paradoxically, FOXO also promotes tumor...
FOXO1 is a transcription factor and potential tumor suppressor that is negatively regulated downstream of PI3K-PKB/AKT signaling. Paradoxically, FOXO also promotes tumor growth, but the detailed mechanisms behind this role of FOXO are not fully understood. In this study, we revealed a molecular cascade by which the Thr24 residue of FOXO1 is phosphorylated by AKT and is dephosphorylated by calcineurin, which is a Ca-dependent protein phosphatase. Curiously, single nucleotide somatic mutations of FOXO1 in cancer occur frequently at and near Thr24. Using a calcineurin inhibitor and shRNA directed against calcineurin, we revealed that calcineurin-mediated dephosphorylation of Thr24 regulates FOXO1 protein stability. We also found that FOXO1 binds to the promoter region of MDM2 and activates transcription, which in turn promotes MDM2-mediated ubiquitination and degradation of p53. FOXO3a and FOXO4 are shown to control p53 activity; however, the significance of FOXO1 in p53 regulation remains largely unknown. Supporting this notion, FOXO1 depletion increased p53 and p21 protein levels in association with the inhibition of cell proliferation. Taken together, these results indicate that FOXO1 is stabilized by calcineurin-mediated dephosphorylation and that FOXO1 supports cancer cell proliferation by promoting MDM2 transcription and subsequent p53 degradation.
Topics: Proto-Oncogene Proteins c-mdm2; Humans; Tumor Suppressor Protein p53; Forkhead Box Protein O1; Calcineurin; Proteolysis; Phosphorylation; Cell Proliferation; Ubiquitination; Cell Line, Tumor; Neoplasms; Forkhead Transcription Factors; Proto-Oncogene Proteins c-akt; Protein Stability
PubMed: 38519029
DOI: 10.1016/j.jbc.2024.107209 -
Experimental Cell Research Apr 2024Plasma saturated free fatty acid (FFA)-induced endothelial dysfunction (ED) contributes to the pathogenesis of atherosclerosis and cardiovascular diseases. However, the...
Plasma saturated free fatty acid (FFA)-induced endothelial dysfunction (ED) contributes to the pathogenesis of atherosclerosis and cardiovascular diseases. However, the mechanism underlying saturated FFA-induced ED remains unclear. This study demonstrated that palmitic acid (PA) induced ED by activating the NADPH oxidase (NOX)/ROS signaling pathway to activate protein phosphatase 4 (PP4) and protein phosphatase 2A (PP2A), thereby reducing endothelial nitric oxide synthase (eNOS) phosphorylation at Ser633 and Ser1177, respectively. Okadaic acid (OA) and fostriecin (FST), which are inhibitors of PP2A, inhibited the PA-induced decreases in eNOS phosphorylation at Ser633 and Ser1177. The antioxidants N-acetylcysteine (NAC) and apocynin (APO) or knockdown of gp91phox or p67phox (NOX subunits) restored PA-mediated downregulation of PP4R2 protein expression and eNOS Ser633 phosphorylation. Knockdown of the PP4 catalytic subunit (PP4c) specifically increased eNOS Ser633 phosphorylation, while silencing the PP2A catalytic subunit (PP2Ac) restored only eNOS Ser1177 phosphorylation. Furthermore, PA dramatically decreased the protein expression of the PP4 regulatory subunit R2 (PP4R2) but not the other regulatory subunits. PP4R2 overexpression increased eNOS Ser633 phosphorylation, nitric oxide (NO) production, cell migration and tube formation but did not change eNOS Ser1177 phosphorylation levels. Coimmunoprecipitation (Co-IP) suggested that PP4R2 and PP4c interacted with the PP4R3α and eNOS proteins. In summary, PA decreases PP4R2 protein expression through the Nox/ROS pathway to activate PP4, which contributes to ED by dephosphorylating eNOS at Ser633. The results of this study suggest that PP4 is a novel therapeutic target for ED and ED-associated vascular diseases.
Topics: Humans; Phosphorylation; Nitric Oxide Synthase Type III; Palmitic Acid; Serine; Reactive Oxygen Species; Cells, Cultured; Protein Phosphatase 2; Vascular Diseases; Nitric Oxide; Phosphoprotein Phosphatases
PubMed: 38513962
DOI: 10.1016/j.yexcr.2024.113998 -
The Journal of Clinical Investigation Mar 2024Fibrosis following tissue injury is distinguished from normal repair by the accumulation of pathogenic and apoptosis-resistant myofibroblasts (MFs), which arise...
Fibrosis following tissue injury is distinguished from normal repair by the accumulation of pathogenic and apoptosis-resistant myofibroblasts (MFs), which arise primarily by differentiation from resident fibroblasts. Endogenous molecular brakes that promote MF dedifferentiation and clearance during spontaneous resolution of experimental lung fibrosis may provide insights that could inform and improve the treatment of progressive pulmonary fibrosis in patients. MAPK phosphatase 1 (MKP1) influences the cellular phenotype and fate through precise and timely regulation of MAPK activity within various cell types and tissues, yet its role in lung fibroblasts and pulmonary fibrosis has not been explored. Using gain- and loss-of-function studies, we found that MKP1 promoted lung MF dedifferentiation and restored the sensitivity of these cells to apoptosis - effects determined to be mainly dependent on MKP1's dephosphorylation of p38α MAPK (p38α). Fibroblast-specific deletion of MKP1 following peak bleomycin-induced lung fibrosis largely abrogated its subsequent spontaneous resolution. Such resolution was restored by treating these transgenic mice with the p38α inhibitor VX-702. We conclude that MKP1 is a critical antifibrotic brake whose inhibition of pathogenic p38α in lung fibroblasts is necessary for fibrosis resolution following lung injury.
Topics: Animals; Mice; Dual Specificity Phosphatase 1; Myofibroblasts; Mitogen-Activated Protein Kinase 14; Pulmonary Fibrosis; Lung; Bleomycin; Humans; Mice, Knockout; Mice, Transgenic; Apoptosis
PubMed: 38512415
DOI: 10.1172/JCI172826 -
Nature Communications Mar 2024Targeting the mitogen-activated protein kinase (MAPK) cascade in pancreatic ductal adenocarcinoma (PDAC) remains clinically unsuccessful. We aim to develop a MAPK...
Targeting the mitogen-activated protein kinase (MAPK) cascade in pancreatic ductal adenocarcinoma (PDAC) remains clinically unsuccessful. We aim to develop a MAPK inhibitor-based therapeutic combination with strong preclinical efficacy. Utilizing a reverse-phase protein array, we observe rapid phospho-activation of human epidermal growth factor receptor 2 (HER2) in PDAC cells upon pharmacological MAPK inhibition. Mechanistically, MAPK inhibitors lead to swift proteasomal degradation of dual-specificity phosphatase 6 (DUSP6). The carboxy terminus of HER2, containing a TEY motif also present in extracellular signal-regulated kinase 1/2 (ERK1/2), facilitates binding with DUSP6, enhancing its phosphatase activity to dephosphorylate HER2. In the presence of MAPK inhibitors, DUSP6 dissociates from the protective effect of the RING E3 ligase tripartite motif containing 21, resulting in its degradation. In PDAC patient-derived xenograft (PDX) models, combining ERK and HER inhibitors slows tumour growth and requires cytotoxic chemotherapy to achieve tumour regression. Alternatively, MAPK inhibitors with trastuzumab deruxtecan, an anti-HER2 antibody conjugated with cytotoxic chemotherapy, lead to sustained tumour regression in most tested PDXs without causing noticeable toxicity. Additionally, KRAS inhibitors also activate HER2, supporting testing the combination of KRAS inhibitors and trastuzumab deruxtecan in PDAC. This study identifies a rational and promising therapeutic combination for clinical testing in PDAC patients.
Topics: Humans; Proto-Oncogene Proteins p21(ras); Protein Kinase Inhibitors; Pancreatic Neoplasms; Carcinoma, Pancreatic Ductal; Mitogen-Activated Protein Kinases; Cell Line, Tumor
PubMed: 38509064
DOI: 10.1038/s41467-024-46811-w