Disease-Causing Mutations in SF3B1 Alter Splicing by Disrupting Interaction with SUGP1.

SF3B1, which encodes an essential spliceosomal protein, is frequently mutated in myelodysplastic syndromes (MDS) and many cancers. However, the defect of mutant SF3B1 is unknown. Here, we analyzed RNA sequencing data from MDS patients and confirmed that SF3B1 mutants use aberrant 3' splice sites. To elucidate the underlying mechanism, we purified complexes containing either wild-type or the hotspot K700E mutant SF3B1 and found that levels of a poorly studied spliceosomal protein, SUGP1, were reduced in mutant spliceosomes. Strikingly, SUGP1 knockdown completely recapitulated the splicing errors, whereas SUGP1 overexpression drove the protein, which our data suggest plays an important role in branchsite recognition, into the mutant spliceosome and partially rescued splicing. Other hotspot SF3B1 mutants showed similar altered splicing and diminished interaction with SUGP1. Our study demonstrates that SUGP1 loss is a common defect of spliceosomes with disease-causing SF3B1 mutations and, because this defect can be rescued, suggests possibilities for therapeutic intervention.

The NFIA-ETO2 fusion blocks erythroid maturation and induces pure erythroid leukemia in cooperation with mutant TP53.

The NFIA-ETO2 fusion is the product of a t(1;16)(p31;q24) chromosomal translocation, so far, exclusively found in pediatric patients with pure erythroid leukemia (PEL). To address the role for the pathogenesis of the disease, we facilitated the expression of the NFIA-ETO2 fusion in murine erythroblasts (EBs). We observed that NFIA-ETO2 significantly increased proliferation and impaired erythroid differentiation of murine erythroleukemia cells and of primary fetal liver-derived EBs. However, NFIA-ETO2-expressing EBs acquired neither aberrant in vitro clonogenic activity nor disease-inducing potential upon transplantation into irradiated syngenic mice. In contrast, in the presence of 1 of the most prevalent erythroleukemia-associated mutations, TP53R248Q, expression of NFIA-ETO2 resulted in aberrant clonogenic activity and induced a fully penetrant transplantable PEL-like disease in mice. Molecular studies support that NFIA-ETO2 interferes with erythroid differentiation by preferentially binding and repressing erythroid genes that contain NFI binding sites and/or are decorated by ETO2, resulting in a activity shift from GATA- to ETS-motif-containing target genes. In contrast, TP53R248Q does not affect erythroid differentiation but provides self-renewal and survival potential, mostly via downregulation of known TP53 targets. Collectively, our work indicates that NFIA-ETO2 initiates PEL by suppressing gene expression programs of terminal erythroid differentiation and cooperates with TP53 mutation to induce erythroleukemia.

FLI1 induces erythroleukemia through opposing effects on UBASH3A and UBASH3B expression.

BACKGROUND: FLI1 is an oncogenic transcription factor that promotes diverse malignancies through mechanisms that are not fully understood. Herein, FLI1 is shown to regulate the expression of Ubiquitin Associated and SH3 Domain Containing A/B (UBASH3A/B) genes. UBASH3B and UBASH3A are found to act as an oncogene and tumor suppressor, respectively, and their combined effect determines erythroleukemia progression downstream of FLI1. METHODS: Promoter analysis combined with luciferase assays and chromatin immunoprecipitation (ChIP) analysis were applied on the UBASH3A/B promoters. RNAseq analysis combined with bioinformatic was used to determine the effect of knocking-down UBASH3A and UBASH3B in leukemic cells. Downstream targets of UBASH3A/B were inhibited in leukemic cells either via lentivirus-shRNAs or small molecule inhibitors. Western blotting and RT-qPCR were used to determine transcription levels, MTT assays to assess proliferation rate, and flow cytometry to examine apoptotic index. RESULTS: Knockdown of FLI1 in erythroleukemic cells identified the UBASH3A/B genes as potential downstream targets. Herein, we show that FLI1 directly binds to the UBASH3B promoter, leading to its activation and leukemic cell proliferation. In contrast, FLI1 indirectly inhibits UBASH3A transcription via GATA2, thereby antagonizing leukemic growth. These results suggest oncogenic and tumor suppressor roles for UBASH3B and UBASH3A in erythroleukemia, respectively. Mechanistically, we show that UBASH3B indirectly inhibits AP1 (FOS and JUN) expression, and that its loss leads to inhibition of apoptosis and acceleration of proliferation. UBASH3B also positively regulates the SYK gene expression and its inhibition suppresses leukemia progression. High expression of UBASH3B in diverse tumors was associated with worse prognosis. In contrast, UBASH3A knockdown in erythroleukemic cells increased proliferation; and this was associated with a dramatic induction of the HSP70 gene, HSPA1B. Accordingly, knockdown of HSPA1B in erythroleukemia cells significantly accelerated leukemic cell proliferation. Accordingly, overexpression of UBASH3A in different cancers was predominantly associated with good prognosis. These results suggest for the first time that UBASH3A plays a tumor suppressor role in part through activation of HSPA1B. CONCLUSIONS: FLI1 promotes erythroleukemia progression in part by modulating expression of the oncogenic UBASH3B and tumor suppressor UBASH3A.

The anti-SARS-CoV-2 BNT162b2 vaccine suppresses mithramycin-induced erythroid differentiation and expression of embryo-fetal globin genes in human erythroleukemia K562 cells.

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is causative of the ongoing coronavirus disease 2019 (COVID-19) pandemic. The SARS-CoV-2 Spike protein (S-protein) plays an important role in the early phase of SARS-CoV-2 infection through efficient interaction with ACE2. The S-protein is produced by RNA-based COVID-19 vaccines, that were fundamental for the reduction of the viral spread within the population and the clinical severity of COVID-19. However, the S-protein has been hypothesized to be responsible for damaging cells of several tissues and for some important side effects of RNA-based COVID-19 vaccines. Considering the impact of COVID-19 and SARS-CoV-2 infection on the hematopoietic system, the aim of this study was to verify the effect of the BNT162b2 vaccine on erythroid differentiation of the human K562 cell line, that has been in the past intensively studied as a model system mimicking some steps of erythropoiesis. In this context, we focused on hemoglobin production and induced expression of embryo-fetal globin genes, that are among the most important features of K562 erythroid differentiation. We found that the BNT162b2 vaccine suppresses mithramycin-induced erythroid differentiation of K562 cells. Reverse-transcription-qPCR and Western blotting assays demonstrated that suppression of erythroid differentiation was associated with sharp inhibition of the expression of α-globin and γ-globin mRNA accumulation. Inhibition of accumulation of ζ-globin and ε-globin mRNAs was also observed. In addition, we provide in silico studies suggesting a direct interaction between SARS-CoV-2 Spike protein and Hb Portland, that is the major hemoglobin produced by K562 cells. This study thus provides information suggesting the need of great attention on possible alteration of hematopoietic parameters following SARS-CoV-2 infection and/or COVID-19 vaccination.

Engineering genetic devices for in vivo control of therapeutic T cell activity triggered by the dietary molecule resveratrol.

Chimeric antigen receptor (CAR)-engineered T cell therapies have been recognized as powerful strategies in cancer immunotherapy; however, the clinical application of CAR-T is currently constrained by severe adverse effects in patients, caused by excessive cytotoxic activity and poor T cell control. Herein, we harnessed a dietary molecule resveratrol (RES)-responsive transactivator and a transrepressor to develop a repressible transgene expression (RESrep) device and an inducible transgene expression (RESind) device, respectively. After optimization, these tools enabled the control of CAR expression and CAR-mediated antitumor function in engineered human cells. We demonstrated that a resveratrol-repressible CAR expression (RESrep-CAR) device can effectively inhibit T cell activation upon resveratrol administration in primary T cells and a xenograft tumor mouse model. Additionally, we exhibit how a resveratrol-inducible CAR expression (RESind-CAR) device can achieve fine-tuned and reversible control over T cell activation via a resveratrol-titratable mechanism. Furthermore, our results revealed that the presence of RES can activate RESind-CAR T cells with strong anticancer cytotoxicity against cells in vitro and in vivo. Our study demonstrates the utility of RESrep and RESind devices as effective tools for transgene expression and illustrates the potential of RESrep-CAR and RESind-CAR devices to enhance patient safety in precision cancer immunotherapies.

Loss of a 7q gene, CUX1, disrupts epigenetically driven DNA repair and drives therapy-related myeloid neoplasms.

Therapy-related myeloid neoplasms (t-MNs) are high-risk late effects with poorly understood pathogenesis in cancer survivors. It has been postulated that, in some cases, hematopoietic stem and progenitor cells (HSPCs) harboring mutations are selected for by cytotoxic exposures and transform. Here, we evaluate this model in the context of deficiency of CUX1, a transcription factor encoded on chromosome 7q and deleted in half of t-MN cases. We report that CUX1 has a critical early role in the DNA repair process in HSPCs. Mechanistically, CUX1 recruits the histone methyltransferase EHMT2 to DNA breaks to promote downstream H3K9 and H3K27 methylation, phosphorylated ATM retention, subsequent γH2AX focus formation and propagation, and, ultimately, 53BP1 recruitment. Despite significant unrepaired DNA damage sustained in CUX1-deficient murine HSPCs after cytotoxic exposures, they continue to proliferate and expand, mimicking clonal hematopoiesis in patients postchemotherapy. As a consequence, preexisting CUX1 deficiency predisposes mice to highly penetrant and rapidly fatal therapy-related erythroleukemias. These findings establish the importance of epigenetic regulation of HSPC DNA repair and position CUX1 as a gatekeeper in myeloid transformation.

Erythroid differentiation in mouse erythroleukemia cells depends on Tmod3-mediated regulation of actin filament assembly into the erythroblast membrane skeleton.

Erythroid differentiation (ED) is a complex cellular process entailing morphologically distinct maturation stages of erythroblasts during terminal differentiation. Studies of actin filament (F-actin) assembly and organization during terminal ED have revealed essential roles for the F-actin pointed-end capping proteins, tropomodulins (Tmod1 and Tmod3). Tmods bind tropomyosins (Tpms), which enhance Tmod capping and F-actin stabilization. Tmods can also nucleate F-actin assembly, independent of Tpms. Tmod1 is present in the red blood cell (RBC) membrane skeleton, and deletion of Tmod1 in mice leads to a mild compensated anemia due to mis-regulated F-actin lengths and membrane instability. Tmod3 is not present in RBCs, and global deletion of Tmod3 leads to embryonic lethality in mice with impaired ED. To further decipher Tmod3's function during ED, we generated a Tmod3 knockout in a mouse erythroleukemia cell line (Mel ds19). Tmod3 knockout cells appeared normal prior to ED, but showed defects during progression of ED, characterized by a marked failure to reduce cell and nuclear size, reduced viability, and increased apoptosis. Tmod3 does not assemble with Tmod1 and Tpms into the Triton X-100 insoluble membrane skeleton during ED, and loss of Tmod3 had no effect on α1,ß1-spectrin and protein 4.1R assembly into the membrane skeleton. However, F-actin, Tmod1 and Tpms failed to assemble into the membrane skeleton during ED in absence of Tmod3. We propose that Tmod3 nucleation of F-actin assembly promotes incorporation of Tmod1 and Tpms into membrane skeleton F-actin, and that this is integral to morphological maturation and cell survival during erythroid terminal differentiation.

TSPO2 translocates 5-aminolevulinic acid into human erythroleukemia cells.

BACKGROUND: 5-Aminolevulinic acid (ALA) is the first precursor of heme biosynthesis pathway. The exogenous addition of ALA to cells leads to protoporphyrin IX (PPIX) accumulation that has been exploited in photodynamic diagnostic and photodynamic therapy. Several types of ALA transporters have been described depending on the cell type, but there was no clear entry pathway for erythroid cells. The 18 kDa translocator protein (TSPO) has been proposed to be involved in the transport of porphyrins and heme analogs. RESULTS: ALA-induced PPIX accumulation in erythroleukemia cells (UT-7 and K562) was impaired by PK 11195, a competitive inhibitor of both transmembrane proteins TSPO (1 and 2). PK 11195 did not modify the activity of the enzymes of heme biosynthesis, suggesting that ALA entry at the plasma membrane was the limiting factor. In contrast, porphobilinogen (PBG)-induced PPIX accumulation was not affected by PK 11195, suggesting that plasma membrane TSPO2 is a selective transporter of ALA. Overexpression of TSPO2 at the plasma membrane of erythroleukemia cells increased ALA-induced PPIX accumulation, confirming the role of TSPO2 in the import of ALA into the cells. CONCLUSIONS: ALA-induced PPIX accumulation in erythroid cells involves TSPO2 as a selective translocator through the plasma membrane. SIGNIFICANCE: This is the first characterisation of molecular mechanisms involving a new actor in ALA transport in ALA-induced PPIX accumulation in erythroleukemia cells, which could be inhibited by specific drug ligands.

Expression Silencing of Glutathione Peroxidase 4 in Mouse Erythroleukemia Cells Delays In Vitro Erythropoiesis.

Among the eight human glutathione peroxidase isoforms, glutathione peroxidase 4 (GPX4) is the only enzyme capable of reducing complex lipid peroxides to the corresponding alcohols. In mice, corruption of the Gpx4 gene leads to embryonic lethality and more detailed expression silencing studies have implicated the enzyme in several physiological processes (e.g., embryonal cerebrogenesis, neuronal function, male fertility). Experiments with conditional knockout mice, in which expression of the Gpx4 gene was silenced in erythroid precursors, indicated a role of Gpx4 in erythropoiesis. To test this hypothesis in a cellular in vitro model we transfected mouse erythroleukemia cells with a Gpx4 siRNA construct and followed the expression kinetics of erythropoietic gene products. Our data indicate that Gpx4 is expressed at high levels in mouse erythroleukemia cells and that expression silencing of the Gpx4 gene delays in vitro erythropoiesis. However, heterozygous expression of a catalytically inactive Gpx4 mutant (Gpx4+/Sec46Ala) did not induce a defective erythropoietic phenotype in different in vivo and ex vivo models. These data suggest that Gpx4 plays a role in erythroid differentiation of mouse erythroleukemia cells but that heterozygous expression of a catalytically inactive Gpx4 is not sufficient to compromise in vivo and ex vivo erythropoiesis.

Identification of Spiro-Fused Pyrrolo[3,4-a]pyrrolizines and Tryptanthrines as Potential Antitumor Agents: Synthesis and In Vitro Evaluation.

A series of heterocyclic compounds containing a spiro-fused pyrrolo[3,4-a]pyrrolizine and tryptanthrin framework have been synthesized and studied as potential antitumor agents. Cytotoxicity of products was screened against human erythroleukemia (K562) and human cervical carcinoma (HeLa) cell lines. Among the screened compounds. 4a, 4b and 5a were active against human erythroleukemia (K562) cell line, while 4a and 5a were active against cervical carcinoma (HeLa) cell line. In agreement with the DNA cytometry studies, the tested compounds have achieved significant cell-cycle perturbation with higher accumulation of cells in G2/M phase and induced apoptosis. Using confocal microscopy, we found that with 4a and 5a treatment of HeLa cells, actin filaments disappeared, and granular actin was distributed diffusely in the cytoplasm in 76-91% of cells. We discovered that HeLa cells after treatment with compounds 4a and 5a significantly reduced the number of cells with filopodium-like membrane protrusions (from 63 % in control cells to 29% after treatment) and a decrease in cell motility.

CRISPR/Cas9 knockouts reveal genetic interaction between strain-transcendent erythrocyte determinants of Plasmodium falciparum invasion.

During malaria blood-stage infections, Plasmodium parasites interact with the RBC surface to enable invasion followed by intracellular proliferation. Critical factors involved in invasion have been identified using biochemical and genetic approaches including specific knockdowns of genes of interest from primary CD34+ hematopoietic stem cells (cRBCs). Here we report the development of a robust in vitro culture system to produce RBCs that allow the generation of gene knockouts via CRISPR/Cas9 using the immortal JK-1 erythroleukemia line. JK-1 cells spontaneously differentiate, generating cells at different stages of erythropoiesis, including terminally differentiated nucleated RBCs that we term "jkRBCs." A screen of small-molecule epigenetic regulators identified several bromodomain-specific inhibitors that promote differentiation and enable production of synchronous populations of jkRBCs. Global surface proteomic profiling revealed that jkRBCs express all known Pfalciparum host receptors in a similar fashion to cRBCs and that multiple Pfalciparum strains invade jkRBCs at comparable levels to cRBCs and RBCs. Using CRISPR/Cas9, we deleted two host factors, basigin (BSG) and CD44, for which no natural nulls exist. BSG interacts with the parasite ligand Rh5, a prominent vaccine candidate. A BSG knockout was completely refractory to parasite invasion in a strain-transcendent manner, confirming the essential role for BSG during invasion. CD44 was recently identified in an RNAi screen of blood group genes as a host factor for invasion, and we show that CD44 knockout results in strain-transcendent reduction in invasion. Furthermore, we demonstrate a functional interaction between these two determinants in mediating Pfalciparum erythrocyte invasion.

Curcumin induces apoptosis in JAK2-mutated cells by the inhibition of JAK2/STAT and mTORC1 pathways.

Myeloproliferative neoplasms are chronic myeloid cancers divided in Philadelphia positive and negative. The JAK2 V617F is the most common mutation in Philadelphia negative patients and results in a constitutive activation of the JAK/STAT pathway, conferring a proliferative advantage and apoptosis inhibition. Recent studies identified a functional crosstalk between the JAK/STAT and mTOR pathways. The identification of an effective therapy is often difficult, so the availability of new therapeutic approaches might be attractive. Previous studies showed that curcumin, the active principle of the Curcuma longa, can suppress JAK2/STAT pathways in different type of cancer and injuries. In this study, we investigated the anti-proliferative and pro-apoptotic effects of curcumin in JAK2 V617F-mutated cells. HEL cell line and cells from patients JAK2 V617F mutated have been incubated with increasing concentrations of curcumin for different time. Apoptosis and proliferation were evaluated. Subsequently, JAK2/STAT and AKT/mTOR pathways were investigated at both RNA and protein levels. We found that curcumin induces apoptosis and inhibition of proliferation in HEL cells. Furthermore, we showed that curcumin inhibits JAK2/STAT and mTORC1 pathways in JAK2 V617F-mutated cells. This inhibition suggests that curcumin could represent an alternative strategy to be explored for the treatment of patients with myeloproliferative neoplasms.

CARM1-mediated methylation of protein arginine methyltransferase 5 represses human γ-globin gene expression in erythroleukemia cells.

Protein arginine methyltransferase 5 (PRMT5) is a member of the arginine methyltransferase protein family that critically mediates the symmetric dimethylation of Arg-3 at histone H4 (H4R3me2s) and is involved in many key cellular processes, including hematopoiesis. However, the post-translational modifications (PTMs) of PRMT5 that may affect its biological functions remain less well-understood. In this study, using MS analyses, we found that PRMT5 itself is methylated in human erythroleukemia Lys-562 cells. Biochemical assays revealed that coactivator-associated arginine methyltransferase 1 (CARM1) interacts directly with and methylates PRMT5 at Arg-505 both in vivo and in vitro. Substitutions at Arg-505 significantly reduced PRMT5's methyltransferase activity, decreased H4R3me2s enrichment at the γ-globin gene promoter, and increased the expression of the γ-globin gene in Lys-562 cells. Moreover, CARM1 knockdown consistently reduced PRMT5 activity and activated γ-globin gene expression. Importantly, we show that CARM1-mediated methylation of PRMT5 is essential for the intracellular homodimerization of PRMT5 to its active form. These results thus reveal a critical PTM of PRMT5 that represses human γ-globin gene expression. We conclude that CARM1-mediated asymmetric methylation of PRMT5 is critical for its dimerization and methyltransferase activity leading to the repression of γ-globin expression. Given PRMT5's crucial role in diverse cellular processes, these findings may inform strategies for manipulating its methyltransferase activity for managing hemoglobinopathy or cancer.

In vitro and in vivo cytotoxic activity and human serum albumin interaction for a methoxy-styryl-thiosemicarbazone.

Thiosemicarbazone is a class of compounds with potential applications in medicine, presenting high capacity to inhibit the growth of cancer cells as well as low toxicity. Because of high interest in anticancer studies involving thiosemicarbazones as new chemotherapeutic agents, a synthetic thiosemicarbazone derivative, 4-N-(2'-methoxy-styryl)-thiosemicarbazone (MTSC) was evaluated in vivo against Ehrlich carcinoma in an animal model. In vivo results demonstrated that MTSC treatment induced the survival of mice and altered significantly the body weight of the surviving mice 12 days after tumor inoculation. Treatment with 30 mg/kg of MTSC exhibited effective cytotoxic activity with T/C values of 150.49% (1 dose) and 278% (2 doses). Its interaction with human serum albumin (HSA), which plays a crucial role in the biodistribution of a wide variety of ligands, was investigated by multiple spectroscopic techniques at 296 K, 303 K, and 310 K, as well as by theoretical calculations. The interaction between HSA and MTSC occurs via ground-state association in the subdomain IIA (Sudlow's site I). The binding is moderate (Ka ≈ 104 M-1), spontaneous, entropically, and enthalpically driven. Molecular docking results suggested hydrogen bonding and hydrophobic interactions as the main binding forces. Overall, the interaction HSA:MTSC could provide therapeutic benefits, improving its cytotoxic efficacy and tolerability.

Multidrug resistance phenotype: Relation between phenotype induction and its characteristics in erythroleukemia cells.

Chemotherapy may be followed by multiple drug resistance (MDR). This is an obstacle in the treatment of cancer. It is therefore essential to understand the mechanisms underlying tumor resistance, especially those involved in the cell target/MDR relationship. To investigate this, the effects of exposing cells to UVB (to target DNA), UVA, and H2 O2 (to target the cell membrane) were observed in K562 (non MDR) and FEPS (MDR) cell lines. The K562 cells were more sensitive to UVA than the FEPS cells. The FEPS cell line was more resistant to H2 O2 than K562, only presenting cytotoxicity 72 h after being exposed to 40 mM, with no ROS increase until 48 h. Both cell lines were sensitive to UVB, presenting cytotoxicity after 24 h, mainly by apoptosis, and showed an increase in ROS levels. Our results indicate that agents acting on DNA may be able to overcome the MDR phenotype.

LDB1-mediated enhancer looping can be established independent of mediator and cohesin.

Mechanistic studies in erythroid cells indicate that LDB1, as part of a GATA1/TAL1/LMO2 complex, brings erythroid-expressed genes into proximity with enhancers for transcription activation. The role of co-activators in establishing this long-range interaction is poorly understood. Here we tested the contributions of the RNA Pol II pre-initiation complex (PIC), mediator and cohesin to establishment of locus control region (LCR)/ß-globin proximity. CRISPR/Cas9 editing of the ß-globin promoter to eliminate the RNA Pol II PIC by deleting the TATA-box resulted in loss of transcription, but enhancer-promoter interaction was unaffected. Additional deletion of the promoter GATA1 site eliminated LDB1 complex and mediator occupancy and resulted in loss of LCR/ß-globin proximity. To separate the roles of LDB1 and mediator in LCR looping, we expressed a looping-competent but transcription-activation deficient form of LDB1 in LDB1 knock down cells: LCR/ß-globin proximity was restored without mediator core occupancy. Further, Cas9-directed tethering of mutant LDB1 to the ß-globin promoter forced LCR loop formation in the absence of mediator or cohesin occupancy. Moreover, ENCODE data and our chromatin immunoprecipitation results indicate that cohesin is almost completely absent from validated and predicted LDB1-regulated erythroid enhancer-gene pairs. Thus, lineage specific factors largely mediate enhancer-promoter looping in erythroid cells independent of mediator and cohesin.

Structural Changes and Aggregation Mechanisms of Two Different Dimers of an IgG2 Monoclonal Antibody.

Protein therapeutics, monoclonal antibodies (mAbs) in particular, are large, structurally complex molecules that are prone to numerous modes of degradation during their production and long-term storage. Physical degradation via protein aggregation is a major concern when developing protein therapeutic candidates for clinical use. A dimer is perhaps the simplest element of protein aggregation, and thus, a better understanding of protein dimers in terms of their structures, intermolecular interactions, and chemical nature will help in the development of rational strategies for reducing aggregation propensity. In this study, two different mAb dimers were generated from an IgG2 monoclonal antibody solution, i.e., a native dimer generated under long-term storage and a thermal dimer from a thermal stress condition. Both IgG2 dimers were characterized in terms of their chemical and physical properties, bioactivity, and conformational dynamics. The native IgG2 dimer was formed mainly through noncovalent association. It displayed minimal differences in biophysical properties and higher-order structure compared to the monomer yet showed compromised in vitro potency, likely because of steric hindrance. In contrast, the thermal IgG2 dimer was mainly disulfide-linked, but even so, no new non-native disulfide bonds were detected by peptide mapping. Two regions within the Fc-CH2 domain of the thermal IgG2 dimer exhibited significantly increased flexibility as measured by hydrogen-deuterium exchange mass spectrometry, and notably, these regions are connected by an intrachain disulfide bond under natively folded conditions. These findings provide a better understanding of dimer formation under long-term storage and thermal stress conditions for this IgG2 mAb, and possible aggregation mechanisms are discussed.

The extreme C-terminal region of kindlin-2 is critical to its regulation of integrin activation.

Kindlin-2 (K2), a 4.1R-ezrin-radixin-moesin (FERM) domain adaptor protein, mediates numerous cellular responses, including integrin activation. The C-terminal 15-amino acid sequence of K2 is remarkably conserved across species but is absent in canonical FERM proteins, including talin. In CHO cells expressing integrin αIIbß3, co-expression of K2 with talin head domain resulted in robust integrin activation, but this co-activation was lost after deletion of as few as seven amino acids from the K2 C terminus. This dependence on the C terminus was also observed in activation of endogenous αIIbß3 in human erythroleukemia (HEL) cells and ß1 integrin activation in macrophage-like RAW264.1 cells. Kindlin-1 (K1) exhibited a similar dependence on its C terminus for integrin activation. Expression of the K2 C terminus as an extension of membrane-anchored P-selectin glycoprotein ligand-1 (PSGL-1) inhibited integrin-dependent cell spreading. Deletion of the K2 C terminus did not affect its binding to the integrin ß3 cytoplasmic tail, but combined biochemical and NMR analyses indicated that it can insert into the F2 subdomain. We suggest that this insertion determines the topology of the K2 FERM domain, and its deletion may affect the positioning of the membrane-binding functions of the F2 subdomain and the integrin-binding properties of its F3 subdomain. Free C-terminal peptide can still bind to K2 and displace the endogenous K2 C terminus but may not restore the conformation needed for integrin co-activation. Our findings indicate that the extreme C terminus of K2 is essential for integrin co-activation and highlight the importance of an atypical architecture of the K2 FERM domain in regulating integrin activation.

Exosomal miR-486 regulates hypoxia-induced erythroid differentiation of erythroleukemia cells through targeting Sirt1.

MicroRNAs (miRNAs) regulate the hypoxia-induced erythroid differentiation of hematopoietic cells. In this study, we identified that miR-486 was a rapid response miRNA to hypoxia in erythroleukemia TF-1 cells. Hypoxia exposure increased both intracellular and miR-486 levels of TF-1 cells. Ectopic miR-486 expression enhanced the growth and erythroid differentiation of TF-1 cells, whereas miR-486 inhibition suppressed their growth and erythroid differentiation. Treatment of TF-1 and cord blood CD34+ cells with exogenous containing miR-486 resulted in an increase of intracellular miR-486 level and enhanced erythroid differentiation. Furthermore, we identified that Sirt1 is a miR-486 target gene which modulates hypoxia-induced erythroid differentiation of TF-1 cells. Thus we identified a novel miRNA regulatory network that contributes to hypoxia-induced erythroid differentiation of hematopoietic cells.

Reduction of Thermotolerance by Heat Shock Protein 90 Inhibitors in Murine Erythroleukemia Cells.

Cells induce heat shock proteins (HSPs) against various stress. However, murine erythroleukemia (MEL) cells do not express HSP72, a heat-inducible member of HSP70 family. So, it is of interest to examine how MEL cells respond to heat stress (44°C, 30 min). Heat stress-induced apoptosis was suppressed by pretreatment of heat shock (44°C, 10 min). Such suppressive effects were maximal at 6 h after heat shock and remained up to 12 h. Interestingly, such effects of heat shock were abrogated by specific inhibitors of HSP90 such as 17-allylamino-17-demethoxygeldanamycin (17-AAG) and novobiocin. From flow cytometric analysis, it was found that MEL cells arrest in G2 phase at 6 h after heat shock, but restore original cell cycle at 12 h. High expression level of HSP90 was maintained before and after heat shock. Phosphorylation of HSP90α was observed in apoptotic cells induced by heat stress, but inhibited by pretreatment of heat shock. Such inhibition was abrogated by 17-AAG. Moreover, c-Jun NH2-terminal kinase (JNK) was activated in heat stress-induced apoptotic cells. Taken together, these results suggest that HSP90α in MEL cells plays an important role in the thermotolerance, i.e., suppression of heat stress-induced apoptosis by heat shock.