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Molecular Cell Jan 2024Ubiquitylation is catalyzed by coordinated actions of E3 and E2 enzymes. Molecular principles governing many important E3-E2 partnerships remain unknown, including those...
Ubiquitylation is catalyzed by coordinated actions of E3 and E2 enzymes. Molecular principles governing many important E3-E2 partnerships remain unknown, including those for RING-family GID/CTLH E3 ubiquitin ligases and their dedicated E2, Ubc8/UBE2H (yeast/human nomenclature). GID/CTLH-Ubc8/UBE2H-mediated ubiquitylation regulates biological processes ranging from yeast metabolic signaling to human development. Here, cryoelectron microscopy (cryo-EM), biochemistry, and cell biology reveal this exquisitely specific E3-E2 pairing through an unconventional catalytic assembly and auxiliary interactions 70-100 Å away, mediated by E2 multisite phosphorylation. Rather than dynamic polyelectrostatic interactions reported for other ubiquitylation complexes, multiple Ubc8/UBE2H phosphorylation sites within acidic CK2-targeted sequences specifically anchor the E2 C termini to E3 basic patches. Positions of phospho-dependent interactions relative to the catalytic domains correlate across evolution. Overall, our data show that phosphorylation-dependent multivalency establishes a specific E3-E2 partnership, is antagonistic with dephosphorylation, rigidifies the catalytic centers within a flexing GID E3-substrate assembly, and facilitates substrate collision with ubiquitylation active sites.
Topics: Humans; Ubiquitin-Conjugating Enzymes; Saccharomyces cerevisiae; Phosphorylation; Cryoelectron Microscopy; Ubiquitin-Protein Ligases; Ubiquitination
PubMed: 38113892
DOI: 10.1016/j.molcel.2023.11.027 -
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 -
IScience Nov 2023DNA damage repair (DDR) is mediated by phosphorylating effectors ATM kinase, CHK2, p53, and γH2AX. We showed earlier that GH suppresses DDR by suppressing pATM,...
DNA damage repair (DDR) is mediated by phosphorylating effectors ATM kinase, CHK2, p53, and γH2AX. We showed earlier that GH suppresses DDR by suppressing pATM, resulting in DNA damage accumulation. Here, we show GH acting through GH receptor (GHR) inducing wild-type p53-inducible phosphatase 1 (WIP1), which dephosphorylated ATM and its effectors in normal human colon cells and three-dimensional human intestinal organoids. Mice bearing GH-secreting xenografts exhibited induced colon WIP1 with suppressed pATM and γH2AX. WIP1 was also induced in buffy coats derived from patients with elevated GH from somatotroph adenomas. In contrast, decreased colon WIP1 was observed in mice. WIP1 inhibition restored ATM phosphorylation and reversed GH-induced DNA damage. We elucidated a novel GH signaling pathway activating Src/AMPK to trigger HIPK2 nuclear-cytoplasmic relocation and suppressing WIP1 ubiquitination. Concordantly, blocking either AMPK or Src abolished GH-induced WIP1. We identify WIP1 as a specific target for GH-mediated epithelial DNA damage accumulation.
PubMed: 37876819
DOI: 10.1016/j.isci.2023.108117 -
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 -
MBio Oct 2023Pyrin, a unique cytosolic receptor, initiates inflammatory responses against RhoA-inactivating bacterial toxins and effectors like YopE and YopT. Understanding pyrin...
Pyrin, a unique cytosolic receptor, initiates inflammatory responses against RhoA-inactivating bacterial toxins and effectors like YopE and YopT. Understanding pyrin regulation is crucial due to its association with dysregulated inflammatory responses, including Familial Mediterranean Fever (FMF), linked to pyrin gene mutations. FMF mutations historically acted as a defense mechanism against plague. Negative regulation of pyrin through PKN phosphorylation is well established, with using the YopM effector to promote pyrin phosphorylation and counteract its activity. This study highlights the importance of phosphoprotein phosphatase activity in positively regulating pyrin inflammasome assembly in phagocytic cells of humans and mice. Oligomeric murine pyrin has S205 phosphorylated before inflammasome assembly, and this study implicates the dephosphorylation of murine pyrin S205 by two catalytic subunits of PP2A in macrophages. These findings offer insights for investigating the regulation of oligomeric pyrin and the balance of kinase and phosphatase activity in pyrin-associated infectious and autoinflammatory diseases.
Topics: Humans; Animals; Mice; Inflammasomes; Pyrin; Protein Processing, Post-Translational; Macrophages; Phosphoprotein Phosphatases; Mutation
PubMed: 37787552
DOI: 10.1128/mbio.02066-23 -
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 -
Biophysics and Physicobiology 2024KaiC is a multifunctional enzyme functioning as the core of the circadian clock system in cyanobacteria: its N-terminal domain has adenosine triphosphatase (ATPase)...
KaiC is a multifunctional enzyme functioning as the core of the circadian clock system in cyanobacteria: its N-terminal domain has adenosine triphosphatase (ATPase) activity, and its C-terminal domain has autokinase and autophosphatase activities targeting own S431 and T432. The coordination of these multiple biochemical activities is the molecular basis for robust circadian rhythmicity. Therefore, much effort has been devoted to elucidating the cooperative relationship between the two domains. However, structural and functional relationships between the two domains remain unclear especially with respect to the dawn phase, at which KaiC relieves its nocturnal history through autodephosphorylation. In this study, we attempted to design a double mutation of S431 and T432 that can capture KaiC as a fully dephosphorylated form with minimal impacts on its structure and function, and investigated the cooperative relationship between the two domains in the night to morning phases from many perspectives. The results revealed that both domains cooperate at the dawn phase through salt bridges formed between the domains, thereby non-locally co-activating two events, ATPase de-inhibition and S431 dephosphorylation. Our further analysis using existing crystal structures of KaiC suggests that the states of both domains are not always in one-to-one correspondence at every phase of the circadian cycle, and their coupling is affected by the interactions with KaiA or adjacent subunits within a KaiC hexamer.
PubMed: 38803331
DOI: 10.2142/biophysico.bppb-v21.0001 -
Maintenance of heme homeostasis in through post-translational regulation of glutamyl-tRNA reductase.Journal of Bacteriology Sep 2023is an important human pathogen responsible for a variety of infections including skin and soft tissue infections, endocarditis, and sepsis. The combination of...
is an important human pathogen responsible for a variety of infections including skin and soft tissue infections, endocarditis, and sepsis. The combination of increasing antibiotic resistance in this pathogen and the lack of an efficacious vaccine underscores the importance of understanding how maintains metabolic homeostasis in a variety of environments, particularly during infection. Within the host, must regulate cellular levels of the cofactor heme to support enzymatic activities without encountering heme toxicity. lutamyl NA eductase (GtrR), the enzyme catalyzing the first committed step in heme synthesis, is an important regulatory node of heme synthesis in Bacteria, Archaea, and Plantae. In many organisms, heme status negatively regulates the abundance of GtrR, controlling flux through the heme synthesis pathway. We identified two residues within GtrR, H32 and R214, that are important for GtrR-heme binding. However, in strains expressing either GtrR or GtrR, heme homeostasis was not perturbed, suggesting an alternative mechanism of heme synthesis regulation occurs in . In this regard, we report that heme synthesis is regulated through phosphorylation and dephosphorylation of GtrR by the serine/threonine kinase Stk1 and the phosphatase Stp1, respectively. Taken together, these results suggest that the mechanisms governing staphylococcal heme synthesis integrate both the availability of heme and the growth status of the cell. IMPORTANCE represents a significant threat to human health. Heme is an iron-containing enzymatic cofactor that can be toxic at elevated levels. During infection, must control heme levels to replicate and survive within the hostile host environment. We identified residues within a heme biosynthetic enzyme that are critical for heme binding however, abrogation of heme binding is not sufficient to perturb heme homeostasis within . This marks a divergence from previously reported mechanisms of heme-dependent regulation of the highly conserved enzyme glutamyl tRNA reductase (GtrR). Additionally, we link cell growth arrest to the modulation of heme levels through the post-translational regulation of GtrR by the kinase Stk1 and the phosphatase Stp1.
Topics: Humans; Heme; Staphylococcus aureus; Bacterial Proteins; Homeostasis; Phosphoric Monoester Hydrolases; Staphylococcal Infections
PubMed: 37655914
DOI: 10.1128/jb.00171-23 -
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