Journal of Biological Chemistry
ISSN / EISSN : 0021-9258 / 1083-351X
Published by: Elsevier BV (10.1016)
Total articles ≅ 274,381
Latest articles in this journal
Journal of Biological Chemistry; https://doi.org/10.1016/j.jbc.2021.101305
CEACAM1-LF, a homotypic cell adhesion adhesion molecule, transduces intracellular signals via a 72 amino acid cytoplasmic domain that contains two immunoreceptor tyrosine based inhibitory motifs (ITIMs) and a binding site for β-catenin. Phosphorylation of Ser503 by PKC in rodent CEACAM1 was shown to affect bile acid transport or hepatosteatosis via the level of ITIM phosphorylation, but the phosphorylation of the equivalent residue in human CEACAM1 (Ser508) was unclear. Here we studied this analogous phosphorylation by NMR analysis of the 15N labeled cytoplasmic domain peptide. Incubation with a variety of Ser/Thr kinases revealed phosphorylation of Ser508 by PKA but not by PKC. The lack of phosphorylation by PKC is likely due to evolutionary sequence changes between the rodent and human genes. Phosphorylation site assignment by mass spectrometry and NMR revealed phosphorylation of Ser472, Ser461 and Ser512 by PKA, of which Ser512 is part of a conserved consensus site for GSK3β binding. We showed here that only after phosphorylation of Ser512 by PKA was GSK3β able to phosphorylate Ser508. Phosphorylation of Ser512 by PKA promoted a tight association with the armadillo repeat domain of β-catenin at an extended region spanning the ITIMs of CEACAM1. The kinetics of phosphorylation of the ITIMs by Src, as well dephosphorylation by SHP2, were affected by the presence of Ser508/512 phosphorylation, suggesting that PKA and GSK3β may regulate the signal transduction activity of human CEACAM1-LF. The interaction of CEACAM1-LF with β-catenin promoted by PKA is suggestive of a tight association between the two ITIMs of CEACAM1-LF.
Journal of Biological Chemistry; https://doi.org/10.1016/j.jbc.2021.101309
Mini-abstract Tau can adopt distinct fibril conformations in different human neurodegenerative diseases which may invoke distinct pathological mechanisms. In a recent issue, Weismiller et al showed that intramolecular disulfide links between cys291 and cys322 for a specific tau isoform containing 4 microtubule-binding repeats direct the formation of a structurally distinct amyloid polymorph. These findings have implications in how oxidative stress can flip switches of tau polymorphism in these disease.
Journal of Biological Chemistry, Volume 297; https://doi.org/10.1016/j.jbc.2021.101285
The above review included an error on the top of page six under the heading “Optical tweezers burst and dwell phases represent the closed/SPr and open/NEx states.” The word “open” was used when “closed” was intended. The corrected sentence should read: “The OT observation that substrate translocation occurs in the burst phase associates the OT kinetic burst phase with the cryo-EM structural closed state.”
Journal of Biological Chemistry; https://doi.org/10.1016/j.jbc.2021.101304
Mint3 is known to enhance aerobic ATP production, known as the Warburg effect, by binding to FIH-1. Since this effect is considered to be beneficial for cancer cells, the interaction is a promising target for cancer therapy. However, previous research has suggested that the interacting region of Mint3 with FIH-1 is intrinsically disordered, which makes investigation of this interaction challenging. Therefore, we adopted thermodynamic and structural studies in solution to clarify the structural and thermodynamical changes of Mint3 binding to FIH-1. First, using a combination of circular dichroism, NMR, and hydrogen/deuterium exchange mass spectrometry (HDX-MS), we confirmed that the N-terminal half, which is the interacting part of Mint3, is mostly disordered. Next, we revealed a large enthalpy and entropy change in the interaction of Mint3 using isothermal titration calorimetry (ITC). The profile is consistent with the model that the flexibility of disordered Mint3 is drastically reduced upon binding to FIH-1. Moreover, we performed a series of ITC experiments with several types of truncated Mint3s, an effective approach since the interacting part of Mint3 is disordered, and identified amino acids 78-88 as a novel core site for binding to FIH-1. The truncation study of Mint3 also revealed the thermodynamic contribution of each part of Mint3 to the interaction with FIH-1, where the core sites contribute to the affinity (ΔG), while other sites only affect enthalpy (ΔH), by forming non-covalent bonds. This insight can serve as a foothold for further investigation of intrinsically disordered regions (IDRs) and drug development for cancer therapy.
Journal of Biological Chemistry; https://doi.org/10.1016/j.jbc.2021.101303
As a central player in the canonical TGF-β signaling pathway, Smad2 transmits the activation of TGF-β receptors at the plasma membrane (PM) to transcriptional regulation in the nucleus. Although it has been well established that binding of TGF-β to its receptors leads to the recruitment and activation of Smad2, the spatiotemporal mechanism by which Smad2 is recruited to the activated TGF-β receptor complex and activated is not fully understood. Here we show that Smad2 selectively and tightly binds phosphatidylinositol-4,5-bisphosphate (PI(4,5)P2) in the PM. The PI(4,5)P2-binding site is located in the MH2 domain that is involved in interaction with the TGF-β receptor I that transduces TGF-β-receptor binding to downstream signaling proteins. Quantitative optical imaging analyses show that PM recruitment of Smad2 is triggered by its interaction with PI(4,5)P2 that is locally enriched near the activated TGF-β receptor complex, leading to its binding to the TGF-β receptor I. The PI(4,5)P2 binding activity of Smad2 is essential for the TGF-β-stimulated phosphorylation, nuclear transport and transcriptional activity of Smad2. Structural comparison of all Smad MH2 domains suggests that membrane lipids may also interact with other Smad proteins and regulate their function in diverse TGF-β-mediated biological processes.
Journal of Biological Chemistry; https://doi.org/10.1016/j.jbc.2021.101302
Cutinases are esterases that release fatty acids from the apoplastic layer in plants. As they accept bulky and hydrophobic substrates, cutinases could be used in many applications, ranging from valorization of bark-rich side streams to plastic recycling. Advancement of these applications with cutinases as biocatalysts, however, requires deeper knowledge of the enzymes' biodiversity and structure-function relationships. Here, we mined over 3000 members from Carbohydrate Esterase family 5 (CE5) for putative cutinases and condensed it to 151 genes from known or putative lignocellulose-targeting organisms. The 151 genes were subjected to a phylogenetic analysis. While cutinases with available crystal structures were phylogenetically closely related, we selected nine phylogenic diverse cutinases for characterization. The nine selected cutinases were recombinantly produced and their kinetic activity was characterized against para-nitrophenol substrates esterified with consecutively longer alkyl chains (pNP-C2 to C16). The investigated cutinases each had a unique activity fingerprint against tested pNP-substrates. The five enzymes with the highest activity on pNP-C12 and C16, indicative of activity on bulky hydrophobic compounds, were selected for in-depth kinetic and structure-function analysis. All five enzymes showed a decrease in kcat values with increasing substrate chain length, while KM values and binding energies (calculated from in silico docking analysis) improved. Two cutinases from Fusarium solani and Cryptococcus sp. exhibited outstandingly low KM values, resulting in high catalytic efficiencies towards pNP-C16. Docking analysis suggested that different clades of the phylogenetic tree may harbor enzymes with different modes of substrate interaction, involving a solvent-exposed catalytic triad, a lipase-like lid, or a clamshell-like active site possibly formed by flexible loops.
Journal of Biological Chemistry; https://doi.org/10.1016/j.jbc.2021.101299
The Sigma-1 receptor (S1R) is a transmembrane protein with important roles in cellular homeostasis in normal physiology and in disease. Especially in neurodegenerative diseases, S1R activation has been shown to provide neuroprotection by modulating calcium signaling, mitochondrial function and reducing endoplasmic reticulum (ER) stress. S1R missense mutations are one of the causes of the neurodegenerative Amyotrophic Lateral Sclerosis and distal hereditary motor neuronopathies. Although the S1R has been studied intensively, basic aspects remain controversial, such as S1R topology and whether it reaches the plasma membrane. To address these questions, we have undertaken several approaches. C-terminal tagging with a small biotin-acceptor peptide and BirA biotinylation in cells suggested a type II membrane orientation (cytosolic N-terminus). However, N-terminal tagging gave an equal probability for both possible orientations. This might explain conflicting reports in the literature, as tags may affect the protein topology. Therefore, we studied untagged S1R using a protease protection assay and a glycosylation mapping approach, introducing N-glycosylation sites. Both methods provided unambiguous results showing that the S1R is a type II membrane protein with a short cytosolic N-terminal tail. Assessments of glycan processing, surface fluorescence-activated cell sorting, and cell surface biotinylation indicated ER-retention, with insignificant exit to the plasma membrane, in the absence or presence of S1R agonists or of ER stress. These findings may have important implications for S1R-based therapeutic approaches.
Journal of Biological Chemistry; https://doi.org/10.1016/j.jbc.2021.101301
Lamin-A/C provides a nuclear scaffold for compartmentalization of genome function that is important for genome integrity. Lamin-A/C dysfunction is associated with cancer, aging, and degenerative diseases. The mechanisms whereby lamin-A/C regulates genome stability remain poorly understood. We demonstrate a crucial role for lamin-A/C in DNA replication. We show that lamin-A/C binds to nascent DNA, especially during replication stress (RS), ensuring the recruitment of replication fork protective factors RPA and RAD51. These ssDNA-binding proteins, considered the first and second responders to RS respectively, function in the stabilization, remodeling, and repair of the stalled fork to ensure proper restart and genome stability. Reduced recruitment of RPA and RAD51 upon lamin-A/C depletion elicits replication fork instability (RFI) characterized by MRE11 nuclease-mediated degradation of nascent DNA, RS-induced DNA damage, and sensitivity to replication inhibitors. Importantly, unlike homologous recombination-deficient cells, RFI in lamin-A/C-depleted cells is not linked to replication fork reversal. Thus, the point of entry of nucleases is not the reversed fork, but regions of ssDNA generated during RS that are not protected by RPA and RAD51. Consistently, RFI in lamin-A/C-depleted cells is rescued by exogenous overexpression of RPA or RAD51. These data unveil involvement of structural nuclear proteins in the protection of ssDNA from nucleases during RS by promoting recruitment of RPA and RAD51 to stalled forks. Supporting this model, we show physical interaction between RPA and lamin-A/C. We suggest that RS is a major source of genomic instability in laminopathies and in lamin-A/C-deficient tumors.
Journal of Biological Chemistry; https://doi.org/10.1016/j.jbc.2021.101300
Highly organized circuits of enteric neurons are required for the regulation of gastrointestinal functions, such as peristaltism or migrating motor complex. However, the factors and molecular mechanisms that regulate the connectivity of enteric neurons and their assembly into functional neuronal networks are largely unknown. A better understanding of the mechanisms by which neurotrophic factors regulate this enteric neuron circuitry is paramount to understanding enteric nervous system (ENS) physiology. EphB2, a receptor tyrosine kinase, is essential for neuronal connectivity and plasticity in the brain, but so far its presence and function in the ENS remains largely unexplored. Here we report that EphB2 is expressed preferentially by enteric neurons relative to glial cells throughout the gut in rats. We show that in primary enteric neurons, activation of EphB2 by its natural ligand ephrinB2 engages ERK signaling pathways. Long-term activation with ephrinB2 decreases EphB2 expression and reduces molecular and functional connectivity in enteric neurons without affecting neuronal density, ganglionic fiber bundles, or overall neuronal morphology. This is highlighted by a loss of neuronal plasticity markers such as synapsin I, PSD95 and synaptophysin, and a decrease of spontaneous miniature synaptic currents. Together, these data identify a critical role for EphB2 in the ENS and reveal a unique EphB2-mediated molecular program of synapse regulation in enteric neurons.
Journal of Biological Chemistry; https://doi.org/10.1016/j.jbc.2021.101291
Metabolic dysfunction is a major driver of tumorigenesis. The serine/threonine kinase mTOR constitutes a key central regulator of metabolic pathways promoting cancer cell proliferation and survival. mTOR activity is regulated by metabolic sensors as well as by numerous factors comprising the PTEN/PI3K/AKT canonical pathway, which are often mutated in cancer. However, some cancers displaying constitutively active mTOR do not carry alterations within this canonical pathway, suggesting alternative modes of mTOR regulation. Since DEPTOR, an endogenous inhibitor of mTOR, was previously found to modulate both mTOR complexe 1 and 2, we investigated the different post-transltionnal modification that could affect its inhibitory function. We found that tyrosine 289 phosphorylation of DEPTOR impairs its interaction with mTOR, leading to increased mTOR activation. Using proximity biotinylation assays, we identified SYK (Spleen tyrosine kinase) as a kinase involved in DEPTOR tyrosine 289 phosphorylation in an ephrin (EPH) receptor-dependent manner. Altogether, our work reveals that phosphorylation of tyrosine 289 of DEPTOR represents a novel molecular switch involved in the regulation of both mTORC1 and mTORC2.