Mechanistic Insights into the Inhibition of a Common CTLA-4 Gene Mutation in the Cytoplasmic Domain.

Cytotoxic T-lymphocyte antigen 4 (CTLA-4) is a pivotal immune checkpoint receptor, playing a crucial role in modulating T-cell activation. In this study, we delved into the underlying mechanism by which a common mutation, G199R, in the cytoplasmic domain of CTLA-4 impacts its inhibitory function. Utilizing nuclear magnetic resonance (NMR) spectroscopy and biochemical techniques, we mapped the conformational changes induced by this mutation and investigated its role in CTLA-4 activity. Our findings reveal that this mutation leads to a distinct conformational alteration, enhancing protein-membrane interactions. Moreover, functional assays demonstrated an improved capacity of the G199R mutant to downregulate T-cell activation, underscoring its potential role in immune-related disorders. These results not only enhance our understanding of CTLA-4 regulatory mechanisms but also provide insights for targeted therapeutic strategies addressing immune dysregulation linked to CTLA-4 mutations.

Trioxacarcin A Interactions with G-Quadruplex DNA Reveal Its Potential New Targets as an Anticancer Agent.

Trioxacarcin (TXN) A was reported to be an anticancer agent through alkylation of dsDNA. G-quadruplex DNA (G4-DNA) is frequently formed in the promoter regions of oncogenes and the ends of telomerase genes, considered as promising drug targets for anticancer therapy. There are no reports about TXN A interactions with G4-DNA. Here, we tested TXN A's interactions with several G4-DNA oligos with parallel, antiparallel, or hybrid folding, respectively. We demonstrated that TXN A preferred to alkylate one flexible guanine in the loops of parallel G4-DNA. The position of the alkylated guanine is in favor of interactions of G4-DNA with TXN A. The structure of TXN A covalently bound RET G4-DNA indicated that TXN A alkylation on RET G4-DNA stabilizes the G4-DNA conformation. These studies opened a new window of how TXN A interacted with G4-DNA, which might hint a new mode of its function as an anticancer agent.

Structural and functional insights into CST tethering in Tetrahymena thermophila telomerase.

Telomerase is crucial for telomere maintenance and genome integrity. The most salient feature of Tetrahymena telomerase is that its CST subcomplex (p75-p45-p19) is tethered to the telomerase catalytic core by interacting with the hub p50. Although the cryoelectron microscopy (cryo-EM) structures of Tetrahymena telomerase have recently been reported, the mechanisms of how and why p50 bridges the CST subcomplex to the telomerase catalytic core remain unclear. Here, we present the nuclear magnetic resonance (NMR) structure of the p75OB1-p50PBM complex. Loss of the interaction between p75 and p50 detaches the CST subcomplex from the telomerase catalytic core in Tetrahymena. The tethering of the CST subcomplex to telomerase is required for telomere homeostasis. However, the detached CST subcomplex is still capable of facilitating the telomeric complementary-strand (C-strand) fill in similar to the non-tethered CST complexes in other organisms. These results expand our understanding of telomere synthesis in Tetrahymena.

Development of charybdotoxin Q18F variant as a selective peptide blocker of neuronal BK(α + ß4) channel for the treatment of epileptic seizures.

Epilepsy is the results from the imbalance between inhibition and excitation in neural circuits, which is mainly treated by some chemical drugs with side effects. Gain-of-function of BK channels or knockout of its ß4 subunit associates with spontaneous epilepsy. Currently, few reports were published about the efficacy of BK(α + ß4) channel modulators in epilepsy prevention. Charybdotoxin is a non-specific inhibitor of BK and other K+ channels. Here, by nuclear magnetic resonance (NMR) and other biochemical techniques, we found that charybdotoxin might interact with the extracellular loop of human ß4 subunit (i.e., hß4-loop) of BK(α + ß4) channel at a molar ratio 4:1 (hß4-loop vs. charybdotoxin). Charybdotoxin enhanced its ability to prevent K+ current of BK(α + ß4 H101Y) channel. The charybdotoxin Q18F variant selectively reduced the neuronal spiking frequency and increased interspike intervals of BK(α + ß4) channel by π-π stacking interactions between its residue Phe18 and residue His101 of hß4-loop. Moreover, intrahippocampal infusion of charybdotoxin Q18F variant significantly increased latency time of seizure, reduced seizure duration and seizure numbers on pentylenetetrazole-induced pre-sensitized rats, inhibited hippocampal hyperexcitability and c-Fos expression, and displayed neuroprotective effects on hippocampal neurons. These results implied that charybdotoxin Q18F variant could be potentially used for intractable epilepsy treatment by therapeutically targeting BK(α + ß4) channel.

Insights into the client protein release mechanism of the ATP-independent chaperone Spy.

Molecular chaperones play a central role in regulating protein homeostasis, and their active forms often contain intrinsically disordered regions (IDRs). However, how IDRs impact chaperone action remains poorly understood. Here, we discover that the disordered N terminus of the prototype chaperone Spy facilitates client release. With NMR spectroscopy and molecular dynamics simulations, we find that the N terminus can bind transiently to the client-binding cavity of Spy primarily through electrostatic interactions mediated by the N-terminal D26 residue. This intramolecular interaction results in a dynamic competition of the N terminus with the client for binding to Spy, which promotes client discharge. Our results reveal the mechanism by which Spy releases clients independent of energy input, thus enriching the current knowledge on how ATP-independent chaperones release their clients and highlighting the importance of synergy between IDRs and structural domains in regulating protein function.

Structural characterization of a dimerization interface in the CD28 transmembrane domain.

CD28 has a crucial role in regulating immune responses by enhancing T cell activation and differentiation. Recent studies have shown that the transmembrane helix (TMH) of CD28 mediates receptor assembly and activity, but a structural characterization of TMH is still lacking. Here, we determined the dimeric helix-helix packing of CD28-TMH using nuclear magnetic resonance (NMR) technology. Unexpectedly, wild-type CD28-TMH alone forms stable tetramers in lipid bicelles instead of dimers. The NMR structure of the CD28-TMH C165F mutant reveals that a GxxxA motif, which is highly conserved in many dimeric assemblies, is located at the dimerization interface. Mutating G160 and A164 can disrupt the transmembrane helix assembly and reduces CD28 enhancement in cells. In contrast, a previously proposed YxxxxT motif does not affect the dimerization of full-length CD28, but it does affect CD28 activity. These results imply that the transmembrane domain of CD28 regulates the signaling transduction in a complicated manner.

NMR Detection and Structural Modeling of the Ethylene Receptor LeETR2 from Tomato.

The gaseous plant hormone ethylene influences many physiological processes in plant growth and development. Plant ethylene responses are mediated by a family of ethylene receptors, in which the N-terminal transmembrane domains are responsible for ethylene binding and membrane localization. Until now, little structural information was available on the molecular mechanism of ethylene responses by the transmembrane binding domain of ethylene receptors. Here, we screened different constructs, fusion tags, detergents, and purification methods of the transmembrane sensor domain of ethylene receptors. However, due to their highly hydrophobic transmembrane domain (TMD), only a KSI-fused LeETR21-131 from tomato yielded a good-quality nuclear magnetic resonance (NMR) spectrum in the organic solvent. Interestingly, a dimer model of LeETR21-131 built by the AlphaFold2 algorithm showed greatly converged structures. The interaction analysis of ethylene and LeETR21-131 using molecular docking and molecular dynamics (MD) simulations demonstrated the potential binding sites of ethylene in LeETR21-131. Our exploration provides valuable knowledge for further understanding of the ethylene-perception process in ethylene receptors.

Prognostic significance of long non coding maternally expressed gene 3 in pediatric acute myeloid leukemia.

ABSTRACT: The purpose of this study was to evaluate the correlation of long non-coding RNA maternally expressed gene 3 (Lnc-MEG3) with disease features, treatment response, and survival in pediatric acute myeloid leukemia (AML) patients.Among 92 de novo pediatric AML patients (before treatment and after 1 course of induction) and 40 controls, bone marrow mononuclear cells were obtained. Then, Lnc-MEG3 expression was determined by reverse transcription quantitative polymerase chain reaction. After 1 course of standard induction therapy of pediatric AML patients, complete remission (CR) was assessed. Furthermore, event-free survival (EFS) and overall survival (OS) were determined according to follow-up data.Lnc-MEG3 was reduced in pediatric AML patients compared with controls. In pediatric AML patients, Lnc-MEG3 was correlated with French-American-Britain subtypes and lower Chinese Medical Association risk stratification, while it was not associated with cytogenetic features, FLT3-ITD mutation, CEBPA mutation, NPM1 mutation, WT1 mutation, or National Comprehensive Cancer Network risk stratification. After 1 course of treatment, Lnc-MEG3 exhibited an up-regulation trend. Furthermore, Lnc-MEG3 was of no difference before treatment between patients with and without CR, while elevated Lnc-MEG3 and change of Lnc-MEG3 after 1 course of treatment were associated with increased CR rate. Additionally, increased Lnc-MEG3 expression before treatment was associated with longer EFS but not OS, while enhanced Lnc-MEG3 expression after 1 course of treatment was correlated with both prolonged EFS and OS.Lnc-MEG3 may have clinical significance as a biomarker for assisting with disease management, treatment optimization, and prognosis improvement in pediatric AML patients.

PD-L1 degradation is regulated by electrostatic membrane association of its cytoplasmic domain.

The cytoplasmic domain of PD-L1 (PD-L1-CD) regulates PD-L1 degradation and stability through various mechanism, making it an attractive target for blocking PD-L1-related cancer signaling. Here, by using NMR and biochemical techniques we find that the membrane association of PD-L1-CD is mediated by electrostatic interactions between acidic phospholipids and basic residues in the N-terminal region. The absence of the acidic phospholipids and replacement of the basic residues with acidic residues abolish the membrane association. Moreover, the basic-to-acidic mutations also decrease the cellular abundance of PD-L1, implicating that the electrostatic interaction with the plasma membrane mediates the cellular levels of PD-L1. Interestingly, distinct from its reported function as an activator of AMPK in tumor cells, the type 2 diabetes drug metformin enhances the membrane dissociation of PD-L1-CD by disrupting the electrostatic interaction, thereby decreasing the cellular abundance of PD-L1. Collectively, our study reveals an unusual regulatory mechanism that controls the PD-L1 level in tumor cells, suggesting an alternative strategy to improve the efficacy of PD-L1-related immunotherapies.

Aha1 Exhibits Distinctive Dynamics Behavior and Chaperone-Like Activity.

Aha1 is the only co-chaperone known to strongly stimulate the ATPase activity of Hsp90. Meanwhile, besides the well-studied co-chaperone function, human Aha1 has also been demonstrated to exhibit chaperoning activity against stress-denatured proteins. To provide structural insights for a better understanding of Aha1's co-chaperone and chaperone-like activities, nuclear magnetic resonance (NMR) techniques were used to reveal the unique structure and internal dynamics features of full-length human Aha1. We then found that, in solution, both the two domains of Aha1 presented distinctive thermal stabilities and dynamics behaviors defined by their primary sequences and three-dimensional structures. The low thermal stability (melting temperature of Aha128-162: 54.45 °C) and the internal dynamics featured with slow motions on the µs-ms time scale were detected for Aha1's N-terminal domain (Aha1N). The aforementioned experimental results suggest that Aha1N is in an energy-unfavorable state, which would therefore thermostatically favor the interaction of Aha1N with its partner proteins such as Hsp90's middle domain. Differently from Aha1N, Aha1C (Aha1's C-terminal domain) exhibited enhanced thermal stability (melting temperature of Aha1204-335: 72.41 °C) and the internal dynamics featured with intermediate motions on the ps-ns time scale. Aha1C's thermal and structural stabilities make it competent for the stabilization of the exposed hydrophobic groove of dimerized Hsp90's N-terminal domain. Of note, according to the NMR data and the thermal shift results, although the very N-terminal region (M1-W27) and the C-terminal relaxin-like factor (RLF) motif showed no tight contacts with the remaining parts of human Aha1, they were identified to play important roles in the recognition of intrinsically disordered pathological α-synuclein.

Inhibitor Development against p7 Channel in Hepatitis C Virus.

Hepatitis C Virus (HCV) is the key cause of chronic and severe liver diseases. The recent direct-acting antiviral agents have shown the clinical success on HCV-related diseases, but the rapid HCV mutations of the virus highlight the sustaining necessity to develop new drugs. p7, the viroporin protein from HCV, has been sought after as a potential anti-HCV drug target. Several classes of compounds, such as amantadine and rimantadine have been testified for p7 inhibition. However, the efficacies of these compounds are not high. Here, we screened some novel p7 inhibitors with amantadine scaffold for the inhibitor development. The dissociation constant (Kd) of 42 ARD-series compounds were determined by nuclear magnetic resonance (NMR) titrations. The efficacies of the two best inhibitors, ARD87 and ARD112, were further confirmed using viral production assay. The binding mode analysis and binding stability for the strongest inhibitor were deciphered by molecular dynamics (MD) simulation. These ARD-series compounds together with 49 previously published compounds were further analyzed by molecular docking. Key pharmacophores were identified among the structure-similar compounds. Our studies suggest that different functional groups are highly correlated with the efficacy for inhibiting p7 of HCV, in which hydrophobic interactions are the dominant forces for the inhibition potency. Our findings provide guiding principles for designing higher affinity inhibitors of p7 as potential anti-HCV drug candidates.

IncRNA MVIH correlates with disease features, predicts treatment response and survival in pediatric acute myeloid leukemia.

OBJECTIVE: Long non-coding RNA microvascular invasion in hepatocellular carcinoma (lnc-MVIH) is correlated with unfavorable prognosis in several malignancies, while limitedly studied in pediatric acute myeloid leukemia (AML). This study aimed to investigate the correlation of lnc-MVIH with disease features, response to induction therapy, and survival in pediatric AML patients. METHODS: A total of 129 de novo pediatric AML patients who were retrospectively analyzed and 60 children with non-malignant hematological diseases who underwent bone marrow examination were reviewed as controls. Bone marrow mononuclear cells (BMMCs) were isolated from all participants to detect lnc-MVIH expression by reverse transcription-quantitative polymerase chain reaction. The complete remission status after 1 course of induction therapy, event-free survival, and overall survival of pediatric AML patients were recorded. RESULTS: Lnc-MVIH was upregulated in pediatric AML patients compared with controls (p < 0.001). In pediatric AML patients, lnc-MVIH was correlated with increased bone marrow blasts, less inv(16) or t(16;16) abnormity, and higher Chinese Medical Association (CMA) risk stratification (all p < 0.05), whereas its correlation with National Comprehensive Cancer Network (NCCN) risk stratification was not statistically significant (p = 0.098). As for prognosis, lnc-MVIH high expression patients presented with lower complete response rate to 1 course of induction therapy (61.5% vs. 79.7%, p = 0.024), shorter event-free survival (median 12.0 months vs. 22.0 months, p = 0.006), and overall survival (median 28.0 months vs. 42.0 months, p = 0.043) compared with lnc-MVIH low expression patients. CONCLUSION: Lnc-MVIH correlates with poor treatment response and unfavorable survival in pediatric AML.

Identification of an allosteric hotspot for additive activation of PPARγ in antidiabetic effects.

Thiazolidinediones (TZDs), such as rosiglitazone (RSG), which activates peroxisome proliferator activated receptor-γ (PPARγ), are a potent class of oral antidiabetic agents with good durability. However, the clinical use of TZDs is challenging because of their side effects, including weight gain and hepatotoxicity. Here, we found that bavachinin (BVC), a lead natural product, additively activates PPARγ with low-dose RSG to preserve the maximum antidiabetic effects while reducing weight gain and hepatotoxicity in db/db mice caused by RSG monotherapy. Structural and biochemical assays demonstrated that an unexplored hotspot around Met329 and Ser332 in helix 5 is triggered by BVC cobinding to RSG-bound PPARγ, thereby allosterically stabilizing the active state of the activation-function 2 motif responsible for additive activation with RSG. Based on this hotspot, we discovered a series of new classes of allosteric agonists inducing the activity of TZDs in the same manner as BVC. Together, our data illustrate that the hotspot of PPARγ is druggable for the discovery of new allosteric synergists, and the combination therapy of allosteric synergists and TZD drugs may provide a potential alternative approach to the treatment of type 2 diabetes mellitus.

Taf14 recognizes a common motif in transcriptional machineries and facilitates their clustering by phase separation.

Saccharomyces cerevisiae TBP associated factor 14 (Taf14) is a well-studied transcriptional regulator that controls diverse physiological processes and that physically interacts with at least seven nuclear complexes in yeast. Despite multiple previous Taf14 structural studies, the nature of its disparate transcriptional regulatory functions remains opaque. Here, we demonstrate that the extra-terminal (ET) domain of Taf14 (Taf14ET) recognizes a common motif in multiple transcriptional coactivator proteins from several nuclear complexes, including RSC, SWI/SNF, INO80, NuA3, TFIID, and TFIIF. Moreover, we show that such partner binding promotes liquid-liquid phase separation (LLPS) of Taf14ET, in a mechanism common to YEATS-associated ET domains (e.g., AF9ET) but not Bromo-associated ET domains from BET-family proteins. Thus, beyond identifying the molecular mechanism by which Taf14ET associates with many transcriptional regulators, our study suggests that Taf14 may function as a versatile nuclear hub that orchestrates transcriptional machineries to spatiotemporally regulate diverse cellular pathways.

Histone H4K20 Demethylation by Two hHR23 Proteins.

Histone methyl groups can be removed by demethylases. Although LSD1 and JmjC domain-containing proteins have been identified as histone demethylases, enzymes for many histone methylation states or sites are still unknown. Here, we perform a screening of a cDNA library containing 2,500 nuclear proteins and identify hHR23A as a histone H4K20 demethylase. Overexpression of hHR23A reduces the levels of H4K20me1/2/3 in cells. In vitro, hHR23A specifically demethylates H4K20me1/2/3 and generates formaldehyde. The enzymatic activity requires Fe(II) and α-ketoglutarate as cofactors and the UBA domains of hHR23A. hHR23B, a protein homologous to hHR23A, also demethylates H4K20me1/2/3 in vitro and in vivo. We further demonstrate that hHR23A/B activate the transcription of coding genes by demethylating H4K20me1 and the transcription of repetitive elements by demethylating H4K20me3. Nuclear magnetic resonance (NMR) analyses demonstrate that an HxxxE motif in the UBA1 domain is crucial for iron binding and demethylase activity. Thus, we identify two hHR23 proteins as histone demethylases.

Backbone 1H, 13C, and 15N resonance assignments of the PRY-SPRY domain of RNF135.

RING finger protein 135 (RNF135, also named Riplet or REUL) exerts multiple biological functions and its C-terminal PRY-SPRY/B30.2 domain is indispensable for most of these functions. RNF135 interacts with RIG-I (retinoic acid-inducible gene-I) via the PRY-SPRY domain and ubiquitinates RIG-I to promote innate anti-viral signaling, while mutations in the RNF135 gene can cause the Macrocephaly, macrosomia, facial dysmorphism (MMFD) syndrome, and RNF135 reportedly regulates the proliferation of glioblastoma cells as well as tongue cancer cells. Nevertheless, structure of full-length RNF135 or its PRY-SPRY domain has not been determined, and structural basis for molecular interactions involving RNF135 is largely unknown. Here we report the backbone 1H, 13C, and 15N chemical shift assignments of the PRY-SPRY domain of RNF135 and the secondary structure elements predicted based on chemical shifts, as well as the perturbations caused by the R286H mutation that is associated with MMFD syndrome. We found that the mutation did not alter the gross structure of the PRY-SPRY domain, so it may have impaired RNF135 function by affecting protein-protein interactions mediated by the domain.

Limited hippocampal neurogenesis in SAMP8 mouse model of Alzheimer's disease.

Increasing adult neurogenesis in the hippocampal formation (HF) has been proposed as a potential foundation for neuronal repair in Alzheimer's disease (AD), but the evidence remains controversial. We used P8 strain of senescence-accelerated mice (SAMP8) as a model of AD to investigate changes in adult neurogenesis. We examined new proliferating cells and their survival in the dentate gyrus (DG) of the HF using 5-bromodeoxyuridine (BrdU) labeling and investigated newborn cell development and differentiation with a combination of phenotype markers. In 5-month-old SAMP8, the number of BrdU(+) cells in the DG was significantly increased relative to controls, in accordance with the rising numbers of doublecortin-positive (DCX(+)) immature neurons. Some of these BrdU(+) cells migrated to cornu ammonis 1 (CA1), possibly related to the compensation of neuronal loss. However, the capacity of neurogenesis to compensate neuronal loss during neurodegeneration was limited. First, only half of the BrdU(+) cells survived 4weeks after mitosis, and even fewer developed into neuron-specific nuclear protein positive (NeuN(+)) mature neurons. Second, the number of BrdU(+) cells and DCX(+) cells was decreased in 10-month-old SAMP8, which exhibited progressive neurodegeneration. In addition, the results provided insight into astrocytes as a crucial component of the neurogenic niche. The number of newborn astrocytes and expression of glial fibrillary acidic protein (GFAP) were diminished in the DG of SAMP8 animals, possibly explaining the insufficient neurogenesis. Thus, stimulating limited neurogenesis in AD by improving the neurogenic niche may have therapeutic potential.