Eradicating leukemic stem cells (LSCs), a major driver of primary resistance and relapse in chronic myeloid leukemia (CML) following tyrosine kinase inhibitor (TKI) treatment, is critical for achieving a cure. Previously, we identified that ANP32B promotes CML LSCs' survival and leukemogenesis by directly binding and inhibiting p53 activity, suggesting a therapeutic opportunity. Here, we show that 1-amino-8-naphthol-2,4-disulfonic acid (ANDS) binds unacetylated p53 and restores p53 activity by disrupting ANP32B-p53 interaction. Consequently, ANDS inhibits CML cell proliferation, impairs LSC function and prolongs survival in the CML mouse model while sparing normal progenitor cells. Based on notion that ANDS, as a previously-identified CaMKP inhibitor, potentially activates p-CaMKIIγ to accelerate CML progression, we find that ANDS combination with KN93, a p-CaMKIIγ inhibitor, synergistically suppresses both TKI-sensitive and -resistant CML. Thus, we highlight that ANDS alone or in combination with CaMKP inhibitor could become a therapeutic strategy for eradicating LSCs and overcoming TKI-resistance in CML.

Gene therapy remains the only cure for von Willebrand disease (VWD), but it is limited by the large von Willebrand factor (VWF) gene size. Variants affecting the VWF propeptide (VWFpp) impair multimerization, causing type 2A/IIC VWD. VWFpp serves as a pH-sensitive template for VWF multimer assembly, suggesting that in trans VWFpp supplementation may restore multimerization in VWF variants with defective propeptides. Co-expression of wild-type VWFpp with mutant full-length VWF in vitro led to modest yet consistent improvements in the VWF multimer profile across eight type 2A/IIC VWD-causing variants. Notably, variants with defect D2:D2 interface required lower levels of VWFpp for multimerization rescue, whereas those with intact D2:D2 interfaces exhibited a greater demand. Furthermore, a transgenic mouse model of type 2A/IIC VWD carrying the p.Tyr87Ser mutation was treated with an AAV9 vector encoding VWFpp under the control of endothelial-specific promoters. VWFpp administration remarkably restored VWF multimerization, increased VWF:CB levels from 15.8% ± 10.2% to 71.2% ± 12.7% for at least 16 weeks, corrected the bleeding tendency and improved platelet function. Both in vitro and in vivo findings demonstrate that in trans VWFpp supplementation can rectify defects in VWF multimerization caused by variants in VWFpp, offering a novel therapeutic strategy for type 2A/IIC VWD.

Neuromedin B receptor (NMBR) is a prototypical class A G protein-coupled receptor (GPCR) that plays a crucial role in histamine-independent itch transmission. However, the mechanisms underlying NMBR's selectivity and activation remain poorly understood. Herein, we utilized multiple replica molecular dynamics simulations, Markov state model (MSM), and community network analysis to explore how NMBR interacts with its two orthosteric ligands namely NMB30 and GRP(14-27) and the distinct activation mechanisms of NMBR. Our findings revealed distinct binding modes for these two ligands, highlighting the significance of the C-terminal dumbbell structure of peptides in ligand positioning and interaction with the "toggle switch" residue W2796.48. Moreover, MSM analysis of the NMBR-GRP(14-27) complex revealed that the active conformation of the receptor appeared for less than half of the simulation time, confirming GRP(14-27) as a weak agonist of NMBR. Additionally, community network analysis identified community 8 as a connector between the orthosteric pocket and the intracellular end of NMBR, suggesting that ligand binding patterns may further influence the extent of receptor activation through allosteric regulation. Collectively, our study not only paves the way for the development of antipruritic therapy, but also provides valuable insights for future studies on GPCR activation.

The central role of angiotensinogen in the control of blood pressure is revealed by a series of crystallographic structures, including complexes with renin. Specifically, the structures provide an understanding of the sequential molecular events that lead to the pre-eclamptic hypertensive crises of pregnancy. The release of the precursor vasopressor peptide from the amino-terminal tail of angiotensinogen appears to be modulated by a redox-sensitive disulphide bridge. Our findings indicate that the activation of the thiol-switch in the circulating maternal angiotensinogen occurs at the placental level in response to oxidative stress, exacerbated by placental insufficiency. We propose here that a contributory factor is the inherent redox stress accompanying the placental exchange of oxygenation between the haemoglobin of the mother (oxy-HbA) and the deoxygenated haemoglobin of the foetus (deoxy-HbF).

Characteristics of the two patient treated with osimertinib.

Abstract Osimertinib is a third-generation covalent EGFR inhibitor that is used in treating non–small cell lung cancer. First-generation EGFR inhibitors were found to elicit pro-differentiation effect on acute myeloid leukemia (AML) cells in preclinical studies, but clinical trials yielded mostly negative results. Here, we report that osimertinib selectively induced apoptosis of CD34+ leukemia stem/progenitor cells but not CD34− cells in EGFR-negative AML and chronic myeloid leukemia (CML). Covalent binding of osimertinib to CD34 at cysteines 199 and 177 and suppression of Src family kinases (SFK) and downstream STAT3 activation contributed to osimertinib-induced cell death. SFK and STAT3 inhibition induced synthetic lethality with osimertinib in primary CD34+ cells. CD34 expression was elevated in AML cells compared with their normal counterparts. Genomic, transcriptomic, and proteomic profiling identified mutation and gene expression signatures of patients with AML with high CD34 expression, and univariate and multivariate analyses indicated the adverse prognostic significance of high expression of CD34. Osimertinib treatment induced responses in AML patient-derived xenograft models that correlated with CD34 expression while sparing normal CD34+ cells. Clinical responses were observed in two patients with CD34high AML who were treated with osimertinib on a compassionate-use basis. These findings reveal the therapeutic potential of osimertinib for treating CD34high AML and CML and describe an EGFR-independent mechanism of osimertinib-induced cell death in myeloid leukemia. Significance: Osimertinib binds CD34 and selectively kills CD34+ leukemia cells to induce remission in preclinical models and patients with AML with a high percentage of CD34+ blasts, providing therapeutic options for myeloid leukemia patients.

General characteristics of AML or CML patient samples used in the experiments.

Objective·To explore the activation methods and activity regulation mechanisms of the testisin zymogen in vitro, laying a foundation for further research on the physiological functions of testisin in organisms. Methods·The eukaryotic expression plasmid for the mouse-derived testisin (mTN) zymogen was constructed through full-gene synthesis and subsequently transfected into eukaryotic HEK293S cells for expression. Purified recombinant testisin protein was obtained through methods such as nickel ion affinity chromatography. Site-directed mutagenesis was performed on the activation site and active center of the zymogen, and the activation mechanism of testisin was analyzed through enzymatic activity assays. Additionally, the activation rate of the protein was determined by altering the incubation conditions of the zymogen. The self-cleavage site of the protease was identified by using Edman degradation and site-directed mutagenesis. Finally, the regulatory mechanism of protease activity was examined by analyzing the proteolytic rate of the protein towards its substrates. Results·The recombinant mTN zymogen was expressed in eukaryotic HEK293S cells, and high-purity, uniform recombinant protein was obtained through a two-step purification process. This protein was found to remain stable under acidic conditions but underwent self-activation in neutral or alkaline environments. The rate of activation was influenced by factors such as pH value and temperature. The self-activation process required on the integrity of the enzyme's activation site, Arg46, and its active center, Ser240. Additionally, the self-activation of the recombinant mTN zymogen was often accompanied by the self-cleavage at the 170/175 loop on the molecular surface, but this self-cleavage did not affect the activity of testisin. Furthermore, the optimal reaction pH for activated mTN was 8.0, with the protein remaining relatively stable near this pH. The optimal reaction temperature was 50 ℃, while the protein was best stored at temperatures below 30 ℃. Zn2+ and Ca2+ were found to significantly inhibit mTN activity. Conclusion·An effective method for the preparation of recombinant mTN zymogen is successfully established, and it is discovered that the recombinant mTN undergoes self-activation and self-cleavage in vitro.

Motif analysis by Scaffold PTM of regulated tyrosine-phosphorylated peptides between vehicle or osimertinib treated CD34+ cells.

Inducing the degradation of KRAS represents a novel strategy to combat cancers with KRAS mutation. In this study, we identify ubiquitin-specific protease 2 (USP2) as a novel deubiquitinating enzyme of KRAS in multiple myeloma (MM). Specifically, we demonstrate that gambogic acid (GA) forms a covalent bond with the Cysteine 284 residue of USP2 through an allosteric pocket, inhibiting its deubiquitinating activity. Inactivation or knockdown of USP2 leads to the degradation of KRAS, resulting in the suppression of MM cell proliferation in vitro and in vivo. Conversely, overexpressing USP2 stabilizes KRAS and partially abrogates GA-induced apoptosis in MM cells. Furthermore, elevated USP2 levels may be associated with poorer prognoses in MM patients. These findings highlight the potential of the USP2/KRAS axis as a therapeutic target in MM, suggesting that strategically inducing KRAS degradation via USP2 inhibition could be a promising approach for treating cancers with KRAS mutations.

Objective·To prepare a serine protease inhibitor (Serpin) derived from Tannerella which is associated with periodontosis, and analyze its specificity in inhibiting target proteases and its structural characteristics. Methods·Through amino acid sequence analysis, a Serpin from the human oral microbiome database (eHOMD) was selected and expressed in Escherichia coli. The recombinant protein was purified using methods such as nickel ion affinity chromatography. Its specificity in inhibiting serine proteases was analyzed, followed by an analysis of its three-dimensional spatial structure using structural biology methods. Results·A novel Serpin, named Tannerpin-M, with methionine as the active center P1 residue, was identified, and a high-purity recombinant protein was successfully prepared from Escherichia coli BL21 (DE3). Further activity testing demonstrated that recombinant Tannerpin-M could effectively form SDS-stable covalent complexes with proteases derived from granulocytes (human neutrophil elastase, cathepsin G, and proteinase 3), as well as with other proteases including kallikrein 1 (KLK1), KLK7, and elastase. Tannerpin-M inhibited KLK7 with a second-order association rate constant of 4.12×104 L/(mol·s). The crystal structure of Tannerpin-M in its relaxed state conformation was resolved at a resolution of 2.4 Å (1 Å=0.1 nm). It revealed that Tannerpin-M possessed a significantly elongated reactive center loop and could undergo the classical conformational transition from a stressed to a relaxed state. Conclusion·Tannerpin-M, derived from oral pathogenic bacteria, is a typical inhibitory Serpin, and can effectively inhibit the serine protease released by granulocytes, by which it may protect the oral pathogenic bacteria from attacks of the human immune system.

Serine protease inhibitor B9 (serpin B9, also known as protease inhibitor 9 or PI9) plays a critical role in regulating the immune response by specifically inhibiting granzyme B, a serine protease found in cytotoxic T lymphocytes and natural killer cells. Despite its potential as an anticancer drug target, the structural details of serpin B9 have remained elusive until now. In this study, a cleaved form of recombinant human serpin B9 was successfully prepared and crystallized. The crystals belonged to space group P212121, with unit-cell parameters a = 68.51, b = 82.32, c = 101.17 Å, and an X-ray diffraction data set was collected at 1.9 Å resolution. The structure shows that serpin B9 adopts a relaxed conformation, with its cleaved reactive-centre loop inserted into the central β-sheet. Unlike other serpins, serpin B9 shows significant structural deviations around helix D, with a larger surface cavity, which could serve as a promising target for small-molecule inhibitors.

PDF file - 329K, Supplementary Figure 1: Mutation of Ring domains of Ring1A/B proteins does not change their subcellular localization. Supplementary Figure 2: qRT-PCR analysis of the known PRC1 target genes HOXC5 and HOXB4 and Snail target gene Cyclin D2 in PanC1-shLuc and PanC1-shRing1A/B cells. Supplementary Figure 3: Simultaneous depletion of Ring1A and Ring1B results in global decrease of H2AK119 monoubiquitination, and Snail loss of repression on E-cadherin in AsPC1 cells.

The sirtuin enzyme family members, SIRT1 and SIRT2, play both tumor-promoting and tumor-suppressing roles, depending on the context and experimental conditions. Compounds that inhibit either SIRT1 or SIRT2 show promising antitumor effects in several types of cancer models, both in vitro and in vivo. The simultaneous inhibition of SIRT1 and SIRT2 is helpful in treating cancer by completely blocking p53 deacetylation, leading to cell death. However, only a few SIRT1/2 dual inhibitors have been developed. Here, we report the discovery of a novel series of SIRT1/2 dual inhibitors via a rational drug design that involved virtual screening and a substructure search. Eleven of the derived compounds exhibited high inhibitory activities, with IC50 < 5 μM and high specificity for both SIRT1 and SIRT2. Compounds hsa55 and PS9 strongly induced apoptosis and showed antiproliferative effects against human leukemia cell lines, which could be due to their ability to increase of p53 and α-tubulin acetylation, as we observed in MOLM-13 cells. Therefore, the new scaffolds of these compounds and their efficacy in leukemia cell lines provide important clues for the further development of novel anti-leukemia drugs.

Extramedullary infiltration (EMI) is a concomitant manifestation that may indicate poor outcome of acute myeloid leukemia (AML). The underlying mechanism remains poorly understood and therapeutic options are limited. Here, we employed single-cell RNA sequencing on bone marrow (BM) and EMI samples from a patient with AML presenting pervasive leukemia cutis. A complement C1Q+ macrophage-like leukemia subset, which was enriched within cutis and existed in BM before EMI manifestations, was identified and further verified in multiple patients with AML. Genomic and transcriptional profiling disclosed mutation and gene expression signatures of patients with EMI that expressed high levels of C1Q. RNA sequencing and quantitative proteomic analysis revealed expression dynamics of C1Q from primary to relapse. Univariate and multivariate analysis demonstrated adverse prognosis significance of C1Q expression. Mechanistically, C1Q expression, which was modulated by transcription factor MAF BZIP transcription factor B, endowed leukemia cells with tissue infiltration ability, which could establish prominent cutaneous or gastrointestinal EMI nodules in patient-derived xenograft and cell line-derived xenograft models. Fibroblasts attracted migration of the C1Q+ leukemia cells through C1Q-globular C1Q receptor recognition and subsequent stimulation of transforming growth factor β1. This cell-to-cell communication also contributed to survival of C1Q+ leukemia cells under chemotherapy stress. Thus, C1Q served as a marker for AML with adverse prognosis, orchestrating cancer infiltration pathways through communicating with fibroblasts and represents a compelling therapeutic target for EMI.

Objective·To prepare a high-purity cyanobacterial serine protease inhibitor, screen its target proteases, and detect its inhibitory activity.Methods·A novel serine protease inhibitor from Arthrospira platensis was identified in the Expanded Human Oral Microbiome Database (eHOMD) by amino acid sequence alignment and named as arthropin. The fusion expression vector pSUMO3-arthropin was constructed and transferred into Escherichia coli (E. coli) BL21(DE3) system for fusion protein expression. The recombinant arthropin was purified by a four-step chromatographic purification approach of nickel affinity chromatography, enzymatic digestion, reverse nickel affinity chromatography, and anion exchange chromatography. In addition, the recombinant arthropin was co-incubated with 14 serine proteases such as activated factor Ⅸ (FⅨa), FⅩa, FⅪa, activated protein C (APC) and kallikrein 1 (KLK1), respectively, and then analyzed by SDS-PAGE. The inhibitory rate of arthropin on KLK1 was assayed with kinetic methods. The crystallization conditions of the recombinant arthropin were screened preliminarily, and the suitable crystals were picked for X-ray diffraction to collect the data. Finally, a sub-stable structure model of arthropin was predicted with AlphaFlod Colab.Results·SDS-PAGE showed that the fused arthropin was successfully expressed in the E. coli BL21(DE3) system, and following purification, the high-purity recombinant arthropin, the relative molecular mass of which was similar to the theoretical value (45 800), was obtained. The co-incubation analysis of recombinant arthropin with 14 serine proteases revealed that arthropin was able to form stable covalent complexes with 9 proteases, including FⅩa, APC, FⅨa, FⅪa, trypsin, cathepsin G, KLK1, KLK7 and thrombin. Arthropin inhibited KLK1 with a second-order association rate constant of 1.7×103 L/(mol·s). Moreover, the recombinant arthropin crystalised under the condition of 25% PEG MME 550, 0.1 mol/L MES (pH 6.5) and 0.01 mol/L ZnCl2 , and the crystals preliminarily diffracted to a resolution of 10 Å (1 Å=1×10-10 m). The analysis of the structure predicted by AlphaFlod Colab revealed that arthropin had the classical structural features of the inhibitory serpin.Conclusion·Arthropin, a serpin from Arthrospira platensis, was successfully obtained with high purity and a broad-spectrum of serine protease inhibition, but at a low inhibitory rate.

Serine protease inhibitors (Serpins) are the most widely distributed protease inhibitors in nature and have been identified from all kingdoms of life. Eukaryotic serpins are most abundant with their activities often subject to modulation by cofactors; however, little is known about the regulation of prokaryotic serpins. To address this, here we prepared a recombinant bacteria serpin, termed chloropin, derived from green sulfur bacteria Chlorobium limicola and solved its crystal structure at 2.2 Å resolution. This showed a canonical inhibitory serpin conformation of native chloropin with a surface-exposed reactive loop and a large central beta-sheet. Enzyme activity analysis showed that chloropin could inhibit multiple proteases, such as thrombin and KLK7 with second order inhibition rate constants at 2.5×10 4 M −1 s −1 and 4.5×10 4 M −1 s −1 respectively, consistent with its P1 arginine residue. Heparin could accelerate the thrombin inhibition by ∼17-fold with a bell-shaped dose-dependent curve as seen with heparin-mediated thrombin inhibition by antithrombin. Interestingly, supercoiled DNA could accelerate the inhibition of thrombin by chloropin by 74-fold, while linear DNA accelerated the reaction by 142-fold through a heparin-like template mechanism. In contrast, DNA did not affect the inhibition of thrombin by antithrombin. These results indicate that DNA is likely a natural modulator of chloropin protecting the cell from endogenous or exogenous environmental proteases, and prokaryotic serpins have diverged during evolution to use different surface subsites for activity modulation.

Dear Editor,Von Willebrand factor (VWF) assembly begins in the endoplasmic reticulum of endothelial cells and megakaryocytes where VWF is synthesized as a precursor with multiple domains (Fig. 1a).The polypeptide including the propeptide and mature VWF chain (proVWF) subsequently forms "tail-to-tail" homodimers through their C-terminal cystine knot (CK) domains.These proVWF dimers are then transported to the Golgi where they assemble into large multimers 'head-to-head' through interchain disulfide bonds between D3 domains of two proVWF dimers 1-3 .The Sadler group firstly identified a peptide containing the Cys 1142 -Cys 1142′ disulfide bond from plasma multimeric VWF 4 .Subsequently, through differential alkylation, proteolytic digestion, and mass spectrometry, they identified two free cysteines Cys 1099 and Cys 1142 from D′D3 monomer and proposed their involvement in D′D3 dimer formation 5 .However, the crystal structure of a monomeric D′D3 mutant (C1099A/ C1142A) reported by Dong and Springer revealed that residue Cys 1099 is largely buried inside of the D′D3 domain and it has to undergo a dramatic conformational change to allow forming a disulfide bond with the same cysteine from the other D′D3 molecule 6,7 .Furthermore, the structures of dimeric D3 domains of Mucin 2 (MUC2), a homologous multimeric protein of VWF, showed Cys 1142 -Cys 1142′ and Cys 1097 -Cys 1097′ disulfide linkages (using VWF residue numbering) 8 .Cys 1091

The coronavirus papain-like protease (PLpro) of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is responsible for viral polypeptide cleavage and the deISGylation of interferon-stimulated gene 15 (ISG15), which enable it to participate in virus replication and host innate immune pathways. Therefore, PLpro is considered an attractive antiviral drug target. Here, we show that parthenolide, a germacrane sesquiterpene lactone, has SARS-CoV-2 PLpro inhibitory activity. Parthenolide covalently binds to Cys-191 or Cys-194 of the PLpro protein, but not the Cys-111 at the PLpro catalytic site. Mutation of Cys-191 or Cys-194 reduces the activity of PLpro. Molecular docking studies show that parthenolide may also form hydrogen bonds with Lys-192, Thr-193, and Gln-231. Furthermore, parthenolide inhibits the deISGylation but not the deubiquitinating activity of PLpro in vitro. These results reveal that parthenolide inhibits PLpro activity by allosteric regulation.