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Cell Reports Aug 2023Mitochondrial morphology is regulated by the post-translational modifications of the dynamin family GTPase proteins including mitofusin 1 (MFN1), MFN2, and...
Mitochondrial morphology is regulated by the post-translational modifications of the dynamin family GTPase proteins including mitofusin 1 (MFN1), MFN2, and dynamin-related protein 1 (DRP1). Mitochondrial phosphatase phosphoglycerate mutase 5 (PGAM5) is emerging as a regulator of these post-translational modifications; however, its precise role in the regulation of mitochondrial morphology is unknown. We show that PGAM5 interacts with MFN2 and DRP1 in a stress-sensitive manner. PGAM5 regulates MFN2 phosphorylation and consequently protects it from ubiquitination and degradation. Further, phosphorylation and dephosphorylation modification of MFN2 regulates its fusion ability. Phosphorylation enhances fission and degradation, whereas dephosphorylation enhances fusion. PGAM5 dephosphorylates MFN2 to promote mitochondrial network formation. Further, using a Drosophila genetic model, we demonstrate that the MFN2 homolog Marf and dPGAM5 are in the same biological pathway. Our results identify MFN2 dephosphorylation as a regulator of mitochondrial fusion and PGAM5 as an MFN2 phosphatase.
Topics: GTP Phosphohydrolases; Phosphoric Monoester Hydrolases; Phosphoglycerate Mutase; Mitochondrial Dynamics; Mitochondrial Proteins; Dynamins
PubMed: 37498743
DOI: 10.1016/j.celrep.2023.112895 -
Immunity Sep 2023Anti-interleukin-17 (IL-17) therapy has been used in various autoimmune diseases. However, the efficacy is unexpectedly limited in several IL-17-associated diseases, and...
Anti-interleukin-17 (IL-17) therapy has been used in various autoimmune diseases. However, the efficacy is unexpectedly limited in several IL-17-associated diseases, and the mechanism of limited efficacy remains unclear. Here, we show that a molecular complex containing the adaptor molecule Act1 and tyrosine phosphatase SHP2 mediated autonomous IL-17R signaling that accelerated and sustained inflammation. SHP2, aberrantly augmented in various autoimmune diseases, was induced by IL-17A itself in astrocytes and keratinocytes, sustaining chemokine production even upon anti-IL-17 therapies. Mechanistically, SHP2 directly interacted with and dephosphorylated Act1, which replaced Act1-TRAF5 complexes and induced IL-17-independent activation of IL-17R signaling. Genetic or pharmacologic inactivation of SHP2, or blocking Act1-SHP2 interaction, paralyzed both IL-17-induced and IL-17-independent signaling and attenuated primary or relapsing experimental autoimmune encephalomyelitis. Therefore, Act1-SHP2 complexes mediate an alternative pathway for autonomous activation of IL-17R signaling, targeting which could be a therapeutic option for IL-17-related diseases in addition to current antibody therapies.
Topics: Animals; Humans; Receptors, Interleukin-17; Adaptor Proteins, Signal Transducing; Inflammation; Encephalomyelitis, Autoimmune, Experimental; Disease Progression
PubMed: 37473759
DOI: 10.1016/j.immuni.2023.06.012 -
Cellular & Molecular Immunology Aug 2023Neutrophil extracellular traps (NETs) participate in the rapid inhibition and clearance of pathogens during infection; however, the molecular regulation of NET formation...
Neutrophil extracellular traps (NETs) participate in the rapid inhibition and clearance of pathogens during infection; however, the molecular regulation of NET formation remains poorly understood. In the current study, we found that inhibition of the wild-type p53-induced phosphatase 1 (Wip1) significantly suppressed the activity of Staphylococcus aureus (S. aureus) and accelerated abscess healing in S. aureus-induced abscess model mice by enhancing NET formation. A Wip1 inhibitor significantly enhanced NET formation in mouse and human neutrophils in vitro. High-resolution mass spectrometry and biochemical assays demonstrated that Coro1a is a substrate of Wip1. Further experiments also revealed that Wip1 preferentially and directly interacts with phosphorylated Coro1a than compared to unphosphorylated inactivated Coro1a. The phosphorylated Ser426 site of Coro1a and the 28-90 aa domain of Wip1 are essential for the direct interaction of Coro1a and Wip1 and for Wip1 dephosphorylation of p-Coro1a Ser426. Wip1 deletion or inhibition in neutrophils significantly upregulated the phosphorylation of Coro1a-Ser426, which activated phospholipase C and subsequently the calcium pathway, the latter of which promoted NET formation after infection or lipopolysaccharide stimulation. This study revealed Coro1a to be a novel substrate of Wip1 and showed that Wip1 is a negative regulator of NET formation during infection. These results support the potential application of Wip1 inhibitors to treat bacterial infections.
Topics: Mice; Humans; Animals; Protein Phosphatase 2C; Extracellular Traps; Abscess; Staphylococcus aureus; Neutrophils; Microfilament Proteins
PubMed: 37386173
DOI: 10.1038/s41423-023-01057-2 -
Molecular Therapy : the Journal of the... Dec 2023Osteoarthritis (OA) is the most common joint disease, but no disease-modifying drugs have been approved for OA treatment. Mitophagy participates in mitochondrial...
Osteoarthritis (OA) is the most common joint disease, but no disease-modifying drugs have been approved for OA treatment. Mitophagy participates in mitochondrial homeostasis regulation by selectively clearing dysfunctional mitochondria, which might contribute to cartilage degeneration in OA. Here, we provide evidence of impaired mitophagy in OA chondrocytes, which exacerbates chondrocyte degeneration. Among the several classic mitophagy-regulating pathways and receptors, we found that FUNDC1 plays a key role in preserving chondrocyte homeostasis by inducing mitophagy. FUNDC1 knockdown in vitro and knockout in vivo decreased mitophagy and exacerbated mitochondrial dysfunction, exacerbating chondrocyte degeneration and OA progression. FUNDC1 overexpression via intra-articular injection of adeno-associated virus alleviated cartilage degeneration in OA. Mechanistically, our study demonstrated that PFKP interacts with and dephosphorylates FUNDC1 to induce mitophagy in chondrocytes. Further analysis identified KD025 as a candidate drug for restoring chondrocyte mitophagy by increasing the FUNDC1-PFKP interaction and thus alleviating cartilage degeneration in mice with DMM-induced OA. Our study highlights the role of the FUNDC1-PFKP interaction in chondrocyte homeostasis via mitophagy induction and identifies KD025 as a promising agent for treating OA by increasing chondrocyte mitophagy.
Topics: Animals; Mice; Mitophagy; Cartilage, Articular; Apoptosis; Osteoarthritis; Chondrocytes; Membrane Proteins; Mitochondrial Proteins
PubMed: 37838829
DOI: 10.1016/j.ymthe.2023.10.016 -
Annual Review of Biochemistry Apr 2024Activating mutations in leucine-rich repeat kinase 2 (LRRK2) represent the most common cause of monogenic Parkinson's disease. LRRK2 is a large multidomain protein... (Review)
Review
Activating mutations in leucine-rich repeat kinase 2 (LRRK2) represent the most common cause of monogenic Parkinson's disease. LRRK2 is a large multidomain protein kinase that phosphorylates a specific subset of the ∼65 human Rab GTPases, which are master regulators of the secretory and endocytic pathways. After phosphorylation by LRRK2, Rabs lose the capacity to bind cognate effector proteins and guanine nucleotide exchange factors. Moreover, the phosphorylated Rabs cannot interact with their cognate prenyl-binding retrieval proteins (also known as guanine nucleotide dissociation inhibitors) and, thus, they become trapped on membrane surfaces. Instead, they gain the capacity to bind phospho-Rab-specific effector proteins, such as RILPL1, with resulting pathological consequences. Rab proteins also act upstream of LRRK2 by controlling its activation and recruitment onto membranes. LRRK2 signaling is counteracted by the phosphoprotein phosphatase PPM1H, which selectively dephosphorylates phospho-Rab proteins. We present here our current understanding of the structure, biochemical properties, and cell biology of LRRK2 and its related paralog LRRK1 and discuss how this information guides the generation of LRRK2 inhibitors for the potential benefit of patients.
PubMed: 38621236
DOI: 10.1146/annurev-biochem-030122-051144 -
Blood Nov 2023The use of Bruton tyrosine kinase inhibitors, such as ibrutinib, to block B-cell receptor signaling has achieved a remarkable clinical response in several B-cell...
The use of Bruton tyrosine kinase inhibitors, such as ibrutinib, to block B-cell receptor signaling has achieved a remarkable clinical response in several B-cell malignancies, including mantle cell lymphoma (MCL) and diffuse large B-cell lymphoma (DLBCL). Acquired drug resistance, however, is significant and affects the long-term survival of these patients. Here, we demonstrate that the transcription factor early growth response gene 1 (EGR1) is involved in ibrutinib resistance. We found that EGR1 expression is elevated in ibrutinib-resistant activated B-cell-like subtype DLBCL and MCL cells and can be further upregulated upon ibrutinib treatment. Genetic and pharmacological analyses revealed that overexpressed EGR1 mediates ibrutinib resistance. Mechanistically, TCF4 and EGR1 self-regulation induce EGR1 overexpression that mediates metabolic reprogramming to oxidative phosphorylation (OXPHOS) through the transcriptional activation of PDP1, a phosphatase that dephosphorylates and activates the E1 component of the large pyruvate dehydrogenase complex. Therefore, EGR1-mediated PDP1 activation increases intracellular adenosine triphosphate production, leading to sufficient energy to enhance the proliferation and survival of ibrutinib-resistant lymphoma cells. Finally, we demonstrate that targeting OXPHOS with metformin or IM156, a newly developed OXPHOS inhibitor, inhibits the growth of ibrutinib-resistant lymphoma cells both in vitro and in a patient-derived xenograft mouse model. These findings suggest that targeting EGR1-mediated metabolic reprogramming to OXPHOS with metformin or IM156 provides a potential therapeutic strategy to overcome ibrutinib resistance in relapsed/refractory DLBCL or MCL.
Topics: Humans; Adult; Animals; Mice; Agammaglobulinaemia Tyrosine Kinase; Oxidative Phosphorylation; Drug Resistance, Neoplasm; Cell Line, Tumor; Antineoplastic Agents; Lymphoma, Mantle-Cell; Lymphoma, Large B-Cell, Diffuse; Metformin; Early Growth Response Protein 1
PubMed: 37738652
DOI: 10.1182/blood.2023020142 -
Cell Death and Differentiation Sep 2023Impaired transcription factor EB (TFEB) function and deficient autophagy activity have been shown to aggravate intervertebral disc (IVD) degeneration (IDD), yet the...
Impaired transcription factor EB (TFEB) function and deficient autophagy activity have been shown to aggravate intervertebral disc (IVD) degeneration (IDD), yet the underlying mechanisms remain less clear. Protein posttranslational modifications (PTMs) are critical for determining TFEB trafficking and transcriptional activity. Here, we demonstrate that TFEB activity is controlled by protein methylation in degenerated nucleus pulposus cells (NPCs), even though TFEB itself is incapable of undergoing methylation. Specifically, protein phosphatase 1 catalytic subunit alpha (PPP1CA), newly identified to dephosphorylate TFEB, contains a K141 mono-methylated site. In degenerated NPCs, increased K141-methylation of PPP1CA disrupts its interaction with TEFB and subsequently blocks TEFB dephosphorylation and nuclear translocation, which eventually leads to autophagy deficiency and NPC senescence. In addition, we found that the PPP1CA-mediated targeting of TFEB is facilitated by the protein phosphatase 1 regulatory subunit 9B (PPP1R9B), which binds with PPP1CA and is also manipulated by K141 methylation. Further proteomic analysis revealed that the protein lysine methyltransferase suppressor of variegation 3-9 homologue 2 (SUV39H2) is responsible for the K141 mono-methylation of PPP1CA. Targeting SUV39H2 effectively mitigates NPC senescence and IDD progression, providing a potential therapeutic strategy for IDD intervention.
Topics: Humans; Methylation; Lysine; Intervertebral Disc Degeneration; Protein Phosphatase 1; Proteomics; Autophagy; Histone-Lysine N-Methyltransferase; Protein Processing, Post-Translational; Basic Helix-Loop-Helix Leucine Zipper Transcription Factors
PubMed: 37605006
DOI: 10.1038/s41418-023-01210-4 -
The Plant Cell Sep 2023Catalase (CAT) is often phosphorylated and activated by protein kinases to maintain hydrogen peroxide (H2O2) homeostasis and protect cells against stresses, but whether...
Catalase (CAT) is often phosphorylated and activated by protein kinases to maintain hydrogen peroxide (H2O2) homeostasis and protect cells against stresses, but whether and how CAT is switched off by protein phosphatases remains inconclusive. Here, we identified a manganese (Mn2+)-dependent protein phosphatase, which we named PHOSPHATASE OF CATALASE 1 (PC1), from rice (Oryza sativa L.) that negatively regulates salt and oxidative stress tolerance. PC1 specifically dephosphorylates CatC at Ser-9 to inhibit its tetramerization and thus activity in the peroxisome. PC1 overexpressing lines exhibited hypersensitivity to salt and oxidative stresses with a lower phospho-serine level of CATs. Phosphatase activity and seminal root growth assays indicated that PC1 promotes growth and plays a vital role during the transition from salt stress to normal growth conditions. Our findings demonstrate that PC1 acts as a molecular switch to dephosphorylate and deactivate CatC and negatively regulate H2O2 homeostasis and salt tolerance in rice. Moreover, knockout of PC1 not only improved H2O2-scavenging capacity and salt tolerance but also limited rice grain yield loss under salt stress conditions. Together, these results shed light on the mechanisms that switch off CAT and provide a strategy for breeding highly salt-tolerant rice.
Topics: Catalase; Oryza; Hydrogen Peroxide; Protein Phosphatase 1; Salt Tolerance; Homeostasis; Plant Proteins
PubMed: 37325884
DOI: 10.1093/plcell/koad167 -
Theranostics 2023As a key endogenous negative regulator of ferroptosis, glutathione peroxidase 4 (GPX4) can regulate its antioxidant function through multiple post-translational...
Protein phosphatase 2A-B55β mediated mitochondrial p-GPX4 dephosphorylation promoted sorafenib-induced ferroptosis in hepatocellular carcinoma via regulating p53 retrograde signaling.
As a key endogenous negative regulator of ferroptosis, glutathione peroxidase 4 (GPX4) can regulate its antioxidant function through multiple post-translational modification pathways. However, the effects of the phosphorylation/dephosphorylation status of GPX4 on the regulation of inducible ferroptosis in hepatocellular carcinoma (HCC) remain unclear. To investigate the effects and molecular mechanism of GPX4 phosphorylation/dephosphorylation modification on ferroptosis in HCC cells. Sorafenib (Sora) was used to establish the ferroptosis model in HCC cells . Using the site-directed mutagenesis method, we generated the mimic GPX4 phosphorylation or dephosphorylation HCC cell lines at specific serine sites of GPX4. The effects of GPX4 phosphorylation/dephosphorylation modification on ferroptosis in HCC cells were examined. The interrelationships among GPX4, p53, and protein phosphatase 2A-B55β subunit (PP2A-B55β) were also explored. To explore the synergistic anti-tumor effects of PP2A activation on Sora-administered HCC, we established PP2A-B55β overexpression xenograft tumors in a nude mice model . In the Sora-induced ferroptosis model of HCC , decreased levels of cytoplasmic and mitochondrial GPX4, mitochondrial dysfunction, and enhanced p53 retrograde signaling occurred under Sora treatment. Further, we found that mitochondrial p53 retrograded remarkably into the nucleus and aggravated Sora-induced ferroptosis. The phosphorylation status of GPX4 at the serine 2 site (GPX4) revealed that mitochondrial p-GPX4 dephosphorylation was positively associated with ferroptosis, and the mechanism might be related to mitochondrial p53 retrograding into the nucleus. In HCC cells overexpressing PP2A-B55β, it was found that PP2A-B55β directly interacted with mitochondrial GPX4 and promoted Sora-induced ferroptosis in HCC. Further, PP2A-B55β reduced the interaction between mitochondrial GPX4 and p53, leading to mitochondrial p53 retrograding into the nucleus. Moreover, it was confirmed that PP2A-B55β enhanced the ferroptosis-mediated tumor growth inhibition and mitochondrial p53 retrograde signaling in the Sora-treated HCC xenograft tumors. Our data uncovered that the PP2A-B55β/p-GPX4/p53 axis was a novel regulatory pathway of Sora-induced ferroptosis. Mitochondrial p-GPX4 dephosphorylation triggered ferroptosis via inducing mitochondrial p53 retrograding into the nucleus, and PP2A-B55β was an upstream signal modulator responsible for mitochondrial p-GPX4 dephosphorylation. Our findings might serve as a potential theranostic strategy to enhance the efficacy of Sora in HCC treatment through the targeted intervention of p-GPX4 dephosphorylation via PP2A-B55β activation.
Topics: Animals; Humans; Mice; Carcinoma, Hepatocellular; Cell Nucleus; Down-Regulation; Drug Resistance, Neoplasm; Ferroptosis; Heterografts; Liver Neoplasms; Mice, Inbred BALB C; Mice, Nude; Mitochondria; Neoplasm Transplantation; Phospholipid Hydroperoxide Glutathione Peroxidase; Phosphorylation; Signal Transduction; Sorafenib; Protein Phosphatase 2
PubMed: 37554285
DOI: 10.7150/thno.82132 -
Circulation Research Oct 2023Atrial fibrillation (AF)-the most common sustained cardiac arrhythmia-increases thromboembolic stroke risk 5-fold. Although atrial hypocontractility contributes to...
BACKGROUND
Atrial fibrillation (AF)-the most common sustained cardiac arrhythmia-increases thromboembolic stroke risk 5-fold. Although atrial hypocontractility contributes to stroke risk in AF, the molecular mechanisms reducing myofilament contractile function remain unknown. We tested the hypothesis that increased expression of PPP1R12C (protein phosphatase 1 regulatory subunit 12C)-the PP1 (protein phosphatase 1) regulatory subunit targeting MLC2a (atrial myosin light chain 2)-causes hypophosphorylation of MLC2a and results in atrial hypocontractility.
METHODS
Right atrial appendage tissues were isolated from human patients with AF versus sinus rhythm controls. Western blots, coimmunoprecipitation, and phosphorylation studies were performed to examine how the PP1c (PP1 catalytic subunit)-PPP1R12C interaction causes MLC2a dephosphorylation. In vitro studies of pharmacological MRCK (myotonic dystrophy kinase-related Cdc42-binding kinase) inhibitor (BDP5290) in atrial HL-1 cells were performed to evaluate PP1 holoenzyme activity on MLC2a. Cardiac-specific lentiviral PPP1R12C overexpression was performed in mice to evaluate atrial remodeling with atrial cell shortening assays, echocardiography, and AF inducibility with electrophysiology studies.
RESULTS
In human patients with AF, PPP1R12C expression was increased 2-fold versus sinus rhythm controls (=2.0×10; n=12 and 12 in each group) with >40% reduction in MLC2a phosphorylation (=1.4×10; n=12 and 12 in each group). PPP1R12C-PP1c binding and PPP1R12C-MLC2a binding were significantly increased in AF (=2.9×10 and 6.7×10, respectively; n=8 and 8 in each group). In vitro studies utilizing drug BDP5290, which inhibits T560-PPP1R12C phosphorylation, demonstrated increased PPP1R12C binding with both PP1c and MLC2a and dephosphorylation of MLC2a. Mice treated with lentiviral PPP1R12C vector demonstrated a 150% increase in left atrial size versus controls (=5.0×10; n=12, 8, and 12), with reduced atrial strain and atrial ejection fraction. Pacing-induced AF in mice treated with lentiviral PPP1R12C vector was significantly higher than in controls (=1.8×10 and 4.1×10, respectively; n=6, 6, and 5).
CONCLUSIONS
Patients with AF exhibit increased levels of PPP1R12C protein compared with controls. PPP1R12C overexpression in mice increases PP1c targeting to MLC2a and causes MLC2a dephosphorylation, which reduces atrial contractility and increases AF inducibility. These findings suggest that PP1 regulation of sarcomere function at MLC2a is a key determinant of atrial contractility in AF.
Topics: Animals; Humans; Mice; Atrial Fibrillation; Heart Atria; Phosphorylation; Protein Phosphatase 1; Stroke
PubMed: 37737016
DOI: 10.1161/CIRCRESAHA.123.322516