Journal of Biological Chemistry最新文献

The protein known as PI3K-interacting protein (PIK3IP1), or transmembrane inhibitor of PI3K (TrIP), is highly expressed by T cells and can modulate PI3K activity in these cells. Several studies have also revealed that TrIP is rapidly downregulated following T cell activation. However, it is unclear how this downregulation is controlled. Using a novel monoclonal antibody that robustly stains cell-surface TrIP, we demonstrate that TrIP is lost from the surface of activated T cells in a manner dependent on the strength of signaling through the T cell receptor and specific downstream signaling pathways, in particular classical PKC isoforms. TrIP expression returns by 24 h after stimulation, suggesting that it may play a role in resetting T cell receptor signaling at later time points. We also provide evidence that ADAM family proteases are required for both constitutive and stimulation-induced downregulation of TrIP in T cells. Finally, by expressing truncated forms of TrIP in cells, we identify the region in the extracellular stalk domain of TrIP that is targeted for proteolytic cleavage.

Cables formed by head-to-tail polymerization of tropomyosin, localized along the length of sarcomeric and cytoskeletal actin filaments, play a key role in regulating a wide range of motile and contractile processes. The stability of tropomyosin cables, their interaction with actin filaments and the functional properties of the resulting co-filaments are thought to be affected by N-terminal acetylation of tropomyosin. Here, we present high-resolution structures of cables formed by acetylated and unacetylated Schizosaccharomyces pombe tropomyosin orthologue Tpm^Cdc8. The crystal structures represent different types of cables, each consisting of Tpm^Cdc8 homodimers in a different conformation. The structures show how the interactions of the residues in the overlap junction contribute to cable formation and how local structural perturbations affect the conformational dynamics of the protein and its ability to transmit allosteric signals. In particular, N-terminal acetylation increases the helicity of the adjacent region, which leads to a local reduction in conformational dynamics and consequently to less fraying of the N-terminal region. This creates a more consistent complementary surface facilitating the formation of specific interactions across the overlap junction.

The pace of the endogenous circadian clock is important for organisms to maintain homeostasis. CHRONO has been shown to be a core component of the mammalian clock and has recently been implicated to function in several important physiological aspects. To function properly, CHRONO needs to enter the nucleus to repress transcription. We have previously shown that the N terminus of CHRONO is required for its nuclear entry. However, how CHRONO enters the nucleus and regulates the circadian clock remains unknown. Here, we report that a novel nonclassical nuclear localization signal in the N terminus of CHRONO is responsible for its nuclear entry. Multiple nuclear transporters are identified that facilitate the nuclear import of CHRONO. We show that the Arg63 is the critical amino acid of the nuclear localization signal. Using prime editing technology, we precisely edit the Arg63 to Ala at the genomic loci and demonstrate that this mutation prolongs the circadian period, which is similar to knockdown of CHRONO. By using the CHRONO KO and R63A mutant cells, we also investigated the changes in the cytoplasmic/nuclear distribution of BMAL1. We show that BMAL1 localizes more in the cytoplasm in the deficiency of CHRONO nuclear entry. These results provide a model for CHRONO nuclear entry using a network of importins involved in the regulation of the circadian period.

The production of human body odor is the result of the action of commensal skin bacteria, including Staphylococcus hominis, acting to biotransform odorless apocrine gland secretions into volatile chemicals like thioalcohols such as 3-methyl-3-sulphanylhexan-1-ol (3M3SH). As the secreted odor precursor Cys-Gly-3M3SH contains a dipeptide, yet the final enzyme in the biotransformation pathway only functions on Cys-3M3SH, we sought to identify the remaining step in this human-adapted biochemical pathway using a novel coupled enzyme assay. Purification of this activity from S. hominis extracts led to the identification of the M20A-family PepV peptidase (ShPepV) as the primary Cys-Gly-3M3SH dipeptidase. To establish whether this was a primary substrate for PepV, the recombinant protein was purified and demonstrated broad activity against diverse dipeptides. The binding site for Cys-Gly-3M3SH was predicted using modeling, which suggested mutations that might accommodate this ligand more favorably. Indeed, a D437A resulted in an almost sixfold increase in the k_cat/K_m, whereas other introduced mutations reduced or abolished function. Together, these data identify an enzyme capable of catalyzing the missing step in an ancient human-specific biochemical transformation and suggest that the production of 3M3SH uses neither a dedicated transporter nor a peptidase for its breakdown, with only the final cleavage step, catalyzed by PatB cysteine-S-conjugate β-lyase, being a unique enzyme.

Neogenin (NEO1) is a ubiquitously expressed transmembrane protein. It interacts with hemojuvelin (HJV). Both NEO1 and HJV play pivotal roles in iron homeostasis by inducing hepcidin expression in the liver. Our previous studies demonstrated that this process depends on Neo1-Hjv interaction and showed that the Hjv-mediated hepcidin expression is correlated with the accumulation of a truncated and membrane-associated form of Neo1. In this study, we tested whether hepcidin expression is induced by increased γ-secretase-mediated cleavage of Neo1 in the liver. We found that Neo1 underwent cleavage of its ectodomain and intracellular domains by α- and γ-secretases, respectively, in hepatoma cells. Our in vitro studies suggest that γ-secretase is responsible for cleavage and release of the cytoplasmic domain of Neo1 in the Hjv-Neo1 complex. This process was enhanced by the inhibition of α-secretase proteolysis and by co-expression with the Neo1-binding partner, Alk3. Further in vivo studies indicated that Neo1 induction of hepcidin expression required γ-secretase cleavage. Interestingly, neither predicted form of γ-secretase-cleaved Neo1 was able to induce hepcidin when separately expressed in hepatocyte-specific Neo1 KO mice. These results imply that the function of Neo1 requires a de novo γ-secretase proteolysis. Additional studies revealed that in addition to the Hjv-binding domains, the function of Neo1 also required its C-terminal intracellular domain and the N-terminal immunoglobulin-like domains that are involved in Neo1 binding to Alk3. Together, our data support the idea that Neo1 induction of hepcidin is initiated as a full-length form and requires a de novo γ-secretase cleavage of Neo1's cytoplasmic domain.

The PII signaling proteins are ubiquitous in prokaryotes serving as crucial metabolic hubs in different metabolic pathways due to their ability to sense and integrate signals of the cellular nitrogen, carbon, and energy levels. In this study we used ligand fishing assays to identify the ribonucleotide monophosphatase UmpH enzyme as a novel target of the PII signaling protein GlnK in Escherichia coli. In vitro analyses showed that UmpH interacts specifically with the PII protein GlnK but not with its paralogue protein GlnB. The UmpH - GlnK complex is modulated by the GlnK uridylylation status and by the levels of the GlnK allosteric effectors ATP, ADP and 2-oxoglutarate. Upon engaging interaction with GlnK, UmpH becomes less active towards its substrate uridine 5'-monophosphate (UMP). We suggest a model where GlnK will physically interact to reduce the UmpH activity during the transition from N-starvation to N-sufficient conditions. Such a mechanism may help the cells to reprogram the fate of UMP from catabolism to anabolism avoiding futile cycling of key nutrients.

The meticulous regulation of intracellular pH (pH_i) is crucial for maintaining cellular function and homeostasis, impacting physiological processes such as heart rhythm, cell migration, proliferation, and differentiation. Dysregulation of pH_i is implicated in various pathologies such as arrhythmias, cancer, and neurodegenerative diseases. Here, we explore the role of STIM1, an ER calcium (Ca²⁺) sensor mediating Store Operated Ca²⁺ Entry (SOCE), in sensing pH_i changes. Our study reveals that STIM1 functions as a sensor for pH_i changes, independent of its Ca²⁺-binding state. Through comprehensive experimental approaches including confocal microscopy, FRET-based sensors, and mutagenesis, we demonstrate that changes in pH_i induce conformational alterations in STIM1, thereby modifying its subcellular localization and activity. We identify two conserved histidine within STIM1 essential for sensing pH_i shifts. Moreover, intracellular alkalization induced by agents such as Angiotensin II or NH₄Cl enhances STIM1-mediated SOCE, promoting cardiac hypertrophy. These findings reveal a novel facet of STIM1 as a multi-modal stress sensor that coordinates cellular responses to both Ca²⁺ and pH fluctuations. This dual functionality underscores its potential as a therapeutic target for diseases associated with pH and Ca²⁺ dysregulation.

Gram-positive bacteria utilize a Fatty Acid Kinase (FAK) complex to harvest fatty acids from the environment. This complex consists of the fatty acid kinase, FakA, and an acyl carrier protein, FakB, and is known to impact virulence and disease outcomes. Despite some recent studies, there remains many outstanding questions as to the enzymatic mechanism and structure of FAK . To better address this gap in knowledge, we used a combination of modeling, biochemical, and cell-based approaches to build on prior proposed models and identify critical details of FAK activity. Using bio-layer interferometry, we demonstrated nanomolar affinity between FakA and FakB that also indicates that FakA is dimer when binding FakB. Additionally, targeted mutagenesis of the FakA Middle domain demonstrates it possesses a metal binding pocket that is critical for FakA dimer stability and FAK function in vitro and in vivo. Lastly, we solved structures of the apo and ligand-bound FakA kinase domain to capture the molecular changes in the protein following ATP binding and hydrolysis. Together, these data provide critical insight into the structure and function of the FAK complex which is essential for understanding its mechanism.

Diterpenoid natural products serve critical functions in plant development and ecological adaptation and many diterpenoids have economic value as bioproducts. The family of class II diterpene synthases catalyzes the committed reactions in diterpenoid biosynthesis, converting a common geranylgeranyl diphosphate precursor into different bicyclic prenyl diphosphate scaffolds. Enzymatic rearrangement and modification of these precursors generate the diversity of bioactive diterpenoids. We report the crystal structure of Grindelia robusta 7,13-copalyl diphosphate synthase, GrTPS2, at 2.1 Å of resolution. GrTPS2 catalyzes the committed reaction in the biosynthesis of grindelic acid, which represents the signature metabolite in species of gumweed (Grindelia spp., Asteraceae). Grindelic acid has been explored as a potential source for drug leads and biofuel production. The GrTPS2 crystal structure adopts the conserved three-domain fold of class II diterpene synthases featuring a functional active site in the γβ-domain and a vestigial ɑ-domain. Substrate docking into the active site of the GrTPS2 apo protein structure predicted catalytic amino acids. Biochemical characterization of protein variants identified residues with impact on enzyme activity and catalytic specificity. Specifically, mutagenesis of Y457 provided mechanistic insight into the position-specific deprotonation of the intermediary carbocation to form the characteristic 7,13 double bond of 7,13-copalyl diphosphate.

We are now in the middle of a so-called 'fourth wave' of drug innovation: multi-specific medicines aimed at diseases and targets previously thought to be "undruggable"; by inducing proximity between two or more proteins, for example a target and an effector that do not naturally interact, such modalities have potential far beyond the scope of conventional drugs. In particular, targeted protein degradation (TPD) strategies to destroy disease-associated proteins have emerged as an exciting pipeline in drug discovery. Most efforts are focused on intracellular proteins, whereas membrane proteins have been less thoroughly explored despite the fact that they comprise roughly a quarter of the human proteome with G-protein coupled receptors (GPCRs) notably dysregulated in many diseases. Here, we discuss the opportunities and the challenges of developing degraders for membrane proteins with a focus on GPCRs. We provide an overview of different TPD platforms in the context of membrane-tethered targets, and we present recent degradation technologies highlighting their potential application to GPCRs.