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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 -
Cancer Letters Apr 2024Gastric cancer (GC) is a common malignant tumor of the digestive tract, and chemoresistance significantly impacts GC patients' prognosis. PANoptosis has been associated...
Gastric cancer (GC) is a common malignant tumor of the digestive tract, and chemoresistance significantly impacts GC patients' prognosis. PANoptosis has been associated with oxaliplatin-induced cell death. However, the direct regulatory role of YBX1 in cellular chemoresistance through PANoptosis remains unclear. In this study, we investigated the impact of YBX1 on regulating PANoptosis and its influence on the resistance of gastric cancer cells to oxaliplatin. Through overexpression and silencing experiments, we assessed YBX1's effect on proliferation and PANoptosis regulation in gastric cancer cells. Additionally, we identified PPM1B and USP10 as interacting proteins with YBX1 and confirmed their influence on YBX1 molecular function and protein expression levels. Our results demonstrate that YBX1 suppresses PANoptosis, leading to enhanced resistance of gastric cancer cells to oxaliplatin. Furthermore, we found that PPM1B and USP10 play critical roles in regulating YBX1-mediated PANoptosis inhibition. PPM1B directly interacts with YBX1, causing dephosphorylation of YBX1 at serine 314 residue. This dephosphorylation process affects the deubiquitination of YBX1 mediated by USP10, resulting in decreased YBX1 protein expression levels and impacting PANoptosis and oxaliplatin resistance in gastric cancer cells. Additionally, we discovered that the 314th amino acid of YBX1 has a profound impact on its own protein expression abundance, thereby affecting the functionality of YBX1. In conclusion, our study reveals the significance of PPM1B-mediated dephosphorylation of YBX1 and USP10-mediated deubiquitination in regulating PANoptosis and sensitivity to oxaliplatin in gastric cancer cells. These findings offer a potential therapeutic strategy for patients with oxaliplatin-resistant gastric cancer.
Topics: Humans; Oxaliplatin; Stomach Neoplasms; Drug Resistance, Neoplasm; Cell Proliferation; Cell Line, Tumor; Gene Expression Regulation, Neoplastic; Ubiquitin Thiolesterase; Y-Box-Binding Protein 1; Protein Phosphatase 2C
PubMed: 38364962
DOI: 10.1016/j.canlet.2024.216712 -
Circulation Research May 2024Endothelial activation promotes the release of procoagulant extracellular vesicles and inflammatory mediators from specialized storage granules. Endothelial membrane...
BACKGROUND
Endothelial activation promotes the release of procoagulant extracellular vesicles and inflammatory mediators from specialized storage granules. Endothelial membrane exocytosis is controlled by phosphorylation. We hypothesized that the absence of PTP1B (protein tyrosine phosphatase 1B) in endothelial cells promotes venous thromboinflammation by triggering endothelial membrane fusion and exocytosis.
METHODS
Mice with inducible endothelial deletion of PTP1B (End.PTP1B-KO) underwent inferior vena cava ligation to induce stenosis and venous thrombosis. Primary endothelial cells from transgenic mice and human umbilical vein endothelial cells were used for mechanistic studies.
RESULTS
Vascular ultrasound and histology showed significantly larger venous thrombi containing higher numbers of Ly6G (lymphocyte antigen 6 family member G)-positive neutrophils in mice with endothelial PTP1B deletion, and intravital microscopy confirmed the more pronounced neutrophil recruitment following inferior vena cava ligation. RT PCR profiler array and immunocytochemistry analysis revealed increased endothelial activation and adhesion molecule expression in primary End.PTP1B-KO endothelial cells, including CD62P (P-selectin) and VWF (von Willebrand factor). Pretreatment with the NF-κB (nuclear factor kappa B) kinase inhibitor BAY11-7082, antibodies neutralizing CD162 (P-selectin glycoprotein ligand-1) or VWF, or arginylglycylaspartic acid integrin-blocking peptides abolished the neutrophil adhesion to End.PTP1B-KO endothelial cells in vitro. Circulating levels of annexin V procoagulant endothelial CD62E (E-selectin) and neutrophil (Ly6G) extracellular vesicles were also elevated in End.PTP1B-KO mice after inferior vena cava ligation. Higher plasma MPO (myeloperoxidase) and Cit-H3 (citrullinated histone-3) levels and neutrophil elastase activity indicated neutrophil activation and extracellular trap formation. Infusion of End.PTP1B-KO extracellular vesicles into C57BL/6J wild-type mice most prominently enhanced the recruitment of endogenous neutrophils, and this response was blunted in VWF-deficient mice or by VWF-blocking antibodies. Reduced PTP1B binding and tyrosine dephosphorylation of SNAP23 (synaptosome-associated protein 23) resulting in increased VWF exocytosis and neutrophil adhesion were identified as mechanisms, all of which could be restored by NF-κB kinase inhibition using BAY11-7082.
CONCLUSIONS
Our findings show that endothelial PTP1B deletion promotes venous thromboinflammation by enhancing SNAP23 phosphorylation, endothelial VWF exocytosis, and neutrophil recruitment.
Topics: Animals; Protein Tyrosine Phosphatase, Non-Receptor Type 1; Exocytosis; Humans; Mice; von Willebrand Factor; Venous Thrombosis; Mice, Knockout; Human Umbilical Vein Endothelial Cells; Inflammation; Mice, Inbred C57BL; Neutrophils; Endothelial Cells; Cells, Cultured; Vena Cava, Inferior; Male; Neutrophil Infiltration; NF-kappa B
PubMed: 38563147
DOI: 10.1161/CIRCRESAHA.124.324214 -
Biochimica Et Biophysica Acta.... Jan 2024Prostate cancer (PC) is the second leading cause of cancer-related death in men. The growth of primary prostate cancer cells relies on circulating androgens and thus the...
Prostate cancer (PC) is the second leading cause of cancer-related death in men. The growth of primary prostate cancer cells relies on circulating androgens and thus the standard therapy for the treatment of localized and advanced PC is the androgen deprivation therapy. Prostatic neuroendocrine carcinoma (PNEC) is an aggressive and highly metastatic subtype of prostate cancer, which displays poor prognosis and high lethality. Most of PNECs develop from prostate adenocarcinoma in response to androgen deprivation therapy, however the mechanisms involved in this transition and in the elevated biological aggressiveness of PNECs are poorly defined. Our current findings indicate that AKAP2 expression is dramatically upregulated in PNECs as compared to non-cancerous prostate tissues. Using a PNEC cell model, we could show that AKAP2 is localized both intracellularly and at the cell periphery where it colocalizes with F-actin. AKAP2 and F-actin interact directly through a newly identified actin-binding domain located on AKAP2. RNAi-mediated silencing of AKAP2 promotes the phosphorylation and deactivation of cofilin, a protein involved in actin turnover. This effect correlates with a significant reduction in cell migration and invasion. Co-immunoprecipitation experiments and proximity ligation assays revealed that AKAP2 forms a complex with the catalytic subunit of protein phosphatase 1 (PP1) in PNECs. Importantly, AKAP2-mediated anchoring of PP1 to the actin cytoskeleton regulates cofilin dephosphorylation and activation, which, in turn, enhances F-actin dynamics and favors migration and invasion. In conclusion, this study identified AKAP2 as an anchoring protein overexpressed in PNECs that controls cancer cell invasive properties by regulating cofilin phosphorylation.
Topics: Male; Humans; A Kinase Anchor Proteins; Actins; Protein Phosphatase 1; Prostatic Neoplasms; Androgen Antagonists; Androgens; Cell Movement; Carcinoma, Neuroendocrine; Actin Depolymerizing Factors; Membrane Proteins
PubMed: 37827203
DOI: 10.1016/j.bbadis.2023.166916 -
IScience Mar 2024The endogenous mechanisms that propagate cardiomyocyte differentiation and prevent de-differentiation remain unclear. While the expression of the heme protein myoglobin...
The endogenous mechanisms that propagate cardiomyocyte differentiation and prevent de-differentiation remain unclear. While the expression of the heme protein myoglobin increases by over 50% during cardiomyocyte differentiation, a role for myoglobin in regulating cardiomyocyte differentiation has not been tested. Here, we show that deletion of myoglobin in cardiomyocyte models decreases the gene expression of differentiation markers and stimulates cellular proliferation, consistent with cardiomyocyte de-differentiation. Mechanistically, the heme prosthetic group of myoglobin catalyzes the oxidation of the Hippo pathway kinase LATS1, resulting in phosphorylation and inactivation of yes-associated protein (YAP). , myoglobin-deficient zebrafish hearts show YAP dephosphorylation and accelerated cardiac regeneration after apical injury. Similarly, myoglobin knockdown in neonatal murine hearts shows increased YAP dephosphorylation and cardiomyocyte cycling. These data demonstrate a novel role for myoglobin as an endogenous driver of cardiomyocyte differentiation and highlight myoglobin as a potential target to enhance cardiac development and improve cardiac repair and regeneration.
PubMed: 38414852
DOI: 10.1016/j.isci.2024.109146 -
Frontiers in Immunology 2023Macrophages play a critical role in the regulation of inflammation and tissue homeostasis. In addition to their vital functions for cell survival and physiology,...
Macrophages play a critical role in the regulation of inflammation and tissue homeostasis. In addition to their vital functions for cell survival and physiology, mitochondria play a crucial role in innate immunity as a platform for the induction of inflammatory responses by regulating cell signaling and dynamics. Dynamin-related protein 1 (Drp1) plays a role in the induction of inflammatory responses and the subsequent development of various diseases. PGAM5 (phosphoglycerate mutase member 5) is a mitochondrial outer membrane phosphatase that dephosphorylates its substrate, Drp1. Previous studies showed that PGAM5 regulates the phosphorylation of Drp1 for the activation of NKT cells and T cells. However, it is not clear how PGAM5 regulates Drp1 activity for the induction of inflammation in macrophages. Here, we demonstrate that PGAM5 activity regulates the dephosphorylation of Drp1 in macrophages, leading to the induction of proinflammatory responses in macrophages. In TLR signaling, PGAM5 regulates the expression and production of inflammatory cytokines by regulating the activation of downstream signaling pathways, including the NF-κB and MAPK pathways. Upon LPS stimulation, PGAM5 interacts with Drp1 to form a complex, leading to the production of mtROS. Furthermore, PGAM5-Drp1 signaling promotes the polarization of macrophages toward a proinflammatory phenotype. Our study further demonstrates that PGAM5-Drp1 signaling promotes metabolic reprogramming by upregulating glycolysis and mitochondrial metabolism in macrophages. Altogether, PGAM5 signaling is a linker between alterations in Drp1-mediated mitochondrial dynamics and inflammatory responses in macrophages and may be a target for the treatment of inflammatory diseases.
Topics: Humans; Dynamins; Inflammation; Macrophages; Mitochondrial Proteins; Phosphoprotein Phosphatases; Signal Transduction; Animals
PubMed: 37771598
DOI: 10.3389/fimmu.2023.1243548 -
International Journal of Molecular... Dec 2023The vertebrate PPP1R15 family consists of the proteins GADD34 (growth arrest and DNA damage-inducible protein 34, the product of the PPP1R15A gene) and CReP... (Review)
Review
The vertebrate PPP1R15 family consists of the proteins GADD34 (growth arrest and DNA damage-inducible protein 34, the product of the PPP1R15A gene) and CReP (constitutive repressor of eIF2α phosphorylation, the product of the PPP1R15B gene), both of which function as targeting/regulatory subunits for protein phosphatase 1 (PP1) by regulating subcellular localization, modulating substrate specificity and assembling complexes with target proteins. The primary cellular function of these proteins is to facilitate the dephosphorylation of eukaryotic initiation factor 2-alpha (eIF2α) by PP1 during cell stress. In this review, we will provide a comprehensive overview of the cellular function, biochemistry and pharmacology of GADD34 and CReP, starting with a brief introduction of eIF2α phosphorylation via the integrated protein response (ISR). We discuss the roles GADD34 and CReP play as feedback inhibitors of the unfolded protein response (UPR) and highlight the critical function they serve as inhibitors of the PERK-dependent branch, which is particularly important since it can mediate cell survival or cell death, depending on how long the stressful stimuli lasts, and GADD34 and CReP play key roles in fine-tuning this cellular decision. We briefly discuss the roles of GADD34 and CReP homologs in model systems and then focus on what we have learned about their function from knockout mice and human patients, followed by a brief review of several diseases in which GADD34 and CReP have been implicated, including cancer, diabetes and especially neurodegenerative disease. Because of the potential importance of GADD34 and CReP in aspects of human health and disease, we will discuss several pharmacological inhibitors of GADD34 and/or CReP that show promise as treatments and the controversies as to their mechanism of action. This review will finish with a discussion of the biochemical properties of GADD34 and CReP, their regulation and the additional interacting partners that may provide insight into the roles these proteins may play in other cellular pathways. We will conclude with a brief outline of critical areas for future study.
Topics: Animals; Humans; Mice; Eukaryotic Initiation Factor-2; Mice, Knockout; Neurodegenerative Diseases; Phosphorylation; Protein Biosynthesis; Protein Phosphatase 1; Proteins
PubMed: 38139150
DOI: 10.3390/ijms242417321 -
BioRxiv : the Preprint Server For... Apr 2024Alpha-synuclein (αsyn) is an intrinsically disordered protein that aggregates in the brain in several neurodegenerative diseases collectively called synucleinopathies....
Alpha-synuclein (αsyn) is an intrinsically disordered protein that aggregates in the brain in several neurodegenerative diseases collectively called synucleinopathies. Phosphorylation of αsyn at serine 129 (PSER129) was considered rare in the healthy human brain but is enriched in pathological αsyn aggregates and is used as a specific marker for disease inclusions. However, recent observations challenge this assumption by demonstrating that PSER129 results from neuronal activity and can be readily detected in the non-diseased mammalian brain. Here, we investigated experimental conditions under which two distinct PSER129 pools, namely endogenous-PSER129 and aggregated-PSER129, could be detected and differentiated in the mammalian brain. Results showed that in the wild-type (WT) mouse brain, perfusion fixation conditions greatly influenced the detection of endogenous-PSER129, with endogenous-PSER129 being nearly undetectable after delayed perfusion fixation (30-minute and 1-hour postmortem interval). Exposure to anesthetics (e.g., Ketamine or xylazine) before perfusion did not significantly influence endogenous-PSER129 detection or levels. In situ, non-specific phosphatase calf alkaline phosphatase (CIAP) selectively dephosphorylated endogenous-PSER129 while αsyn preformed fibril (PFF)-seeded aggregates and genuine disease aggregates (Lewy pathology and Papp-Lantos bodies in Parkinson's disease and multiple systems atrophy brain, respectively) were resistant to CIAP-mediated dephosphorylation. The phosphatase resistance of aggregates was abolished by sample denaturation, and CIAP-resistant PSER129 was closely associated with proteinase K (PK)-resistant αsyn (i.e., a marker of aggregation). CIAP pretreatment allowed for highly specific detection of seeded αsyn aggregates in a mouse model that accumulates non-aggregated-PSER129. We conclude that αsyn aggregates are impervious to phosphatases, and CIAP pretreatment increases detection specificity for aggregated-PSER129, particularly in well-preserved biological samples (e.g., perfusion fixed or flash-frozen mammalian tissues) where there is a high probability of interference from endogenous-PSER129. Our findings have important implications for the mechanism of PSER129-accumulation in the synucleinopathy brain and provide a simple experimental method to differentiate endogenous-from aggregated PSER129.
PubMed: 38645137
DOI: 10.1101/2023.11.20.567854 -
BioRxiv : the Preprint Server For... Jul 2023Jansen de Vries Syndrome (JdVS) is a rare neurodevelopmental disorder (NDD) caused by gain-of-function (GOF) truncating mutations in exons 5 or 6. PPM1D is a...
BACKGROUND
Jansen de Vries Syndrome (JdVS) is a rare neurodevelopmental disorder (NDD) caused by gain-of-function (GOF) truncating mutations in exons 5 or 6. PPM1D is a serine/threonine phosphatase that plays an important role in the DNA damage response (DDR) by negatively regulating TP53 (P53). JdVS-associated mutations lead to the formation of a truncated PPM1D protein that retains catalytic activity and has a GOF effect because of reduced degradation. Somatic exons 5 and 6 truncating mutations are well-established factors in a number of cancers, due to excessive dephosphorylation and reduced function of P53 and other substrates involved in DDR. Children with JdVS have a variety of neurodevelopmental, psychiatric, and physical problems. In addition, a small fraction has acute neuropsychiatric decompensation apparently triggered by infection or severe non-infectious environmental stress factors.
METHODS
To understand the molecular basis of JdVS, we developed an induced pluripotent stem cell (iPSC) model system. iPSCs heterozygous for the truncating variant (), were made from a patient, and control lines engineered using CRISPR-Cas9 gene editing. Proteomics and phosphoprotemics analyses were carried out on iPSC-derived glutamatergic neurons and microglia from three control and three iPSC lines. We also analyzed the effect of the TLR4 agonist, lipopolysaccharide, to understand how activation of the innate immune system in microglia could account for acute behavioral decompensation.
RESULTS
One of the major findings was the downregulation of POGZ in unstimulated microglia. Since loss-of-function variants in the gene are well-known causes of autism spectrum disorder, the decrease in microglia suggests this plays a role in the neurodevelopmental aspects of JdVS. In addition, neurons, baseline, and LPS-stimulated microglia show marked alterations in the expression of several E3 ubiquitin ligases, most notably UBR4, and regulators of innate immunity, chromatin structure, ErbB signaling, and splicing. In addition, pathway analysis points to overlap with neurodegenerative disorders.
LIMITATIONS
Owing to the cost and labor-intensive nature of iPSC research, the sample size was small.
CONCLUSIONS
Our findings provide insight into the molecular basis of JdVS and can be extrapolated to understand neuropsychiatric decompensation that occurs in subgroups of patients with ASD and other NDDs.
PubMed: 37461463
DOI: 10.1101/2023.07.08.548192 -
BioRxiv : the Preprint Server For... Aug 2023Progression through the cell cycle is controlled by regulated and abrupt changes in phosphorylation. Mitotic entry is initiated by increased phosphorylation of mitotic...
Progression through the cell cycle is controlled by regulated and abrupt changes in phosphorylation. Mitotic entry is initiated by increased phosphorylation of mitotic proteins, a process driven by kinases, while mitotic exit is achieved by counteracting dephosphorylation, a process driven by phosphatases, especially PP2A:B55. While the role of kinases in mitotic entry is well-established, recent data have shown that mitosis is only successfully initiated when the counterbalancing phosphatases are also inhibited. For PP2A:B55, inhibition is achieved by the two intrinsically disordered proteins (IDPs), ARPP19 (phosphorylation-dependent) and FAM122A (inhibition is phosphorylation-independent). Despite their critical roles in mitosis, the mechanisms by which they achieve PP2A:B55 inhibition is unknown. Here, we report the cryo-electron microscopy structures of PP2A:B55 bound to phosphorylated ARPP19 and FAM122A. Consistent with our complementary NMR spectroscopy studies both IDPs bind PP2A:B55, but do so in highly distinct manners, unexpectedly leveraging multiple distinct binding sites on B55. Our extensive structural, biophysical and biochemical data explain how substrates and inhibitors are recruited to PP2A:B55 and provides a molecular roadmap for the development of therapeutic interventions for PP2A:B55 related diseases.
PubMed: 37693408
DOI: 10.1101/2023.08.31.555365