RNA-Seq Analysis Reveals Altered Expression of Cell Adhesion-Related Genes Following PZR Knockout in Lung Cancer Cells.

Protein zero related (PZR) serves as a substrate and anchor protein for SHP-2, the product of the proto-oncogene PTPN11 that is frequently mutated in cancers. The expression level of PZR is elevated in various cancers, which is correlated with an unfavorable prognosis. The role of PZR in lung cancer is not fully studied. To investigate how PZR affects signaling pathways involved in LUAD development, we utilized the CRISPR technology to knock out PZR expression in SPC-A1 lung adenocarcinoma cells and then conducted RNA sequencing to profile the transcriptome. Our results showed that 226 genes exhibited differential expressions in PZR-knockout SPC-A1 cells vs wild-type cells. Many of the genes encode proteins involved in cell adhesion, migration, actin cytoskeleton organization, and regulation of cell shape. Furthermore, our experimental data showed that PZR-knockout SPC-A1 cells displayed faster attachment to tissue culture dishes and slower detachment from the dishes upon EDTA treatment. The data suggest an important role of PZR in cell-matrix interaction and may provide new insights into the signaling events that regulate cancer development.

miR-221/222 induce instability of p53 By downregulating deubiquitinase YOD1 in acute myeloid leukemia.

Acute myeloid leukemia (AML) is a hematological malignancy characterized by the impaired differentiation and uncontrolled proliferation of myeloid blasts. Tumor suppressor p53 is often downregulated in AML cells via ubiquitination-mediated degradation. While the role of E3 ligase MDM2 in p53 ubiquitination is well-accepted, little is known about the involvement of deubiquitinases (DUBs). Herein, we found that the expression of YOD1, among several DUBs, is substantially reduced in blood cells from AML patients. We identified that YOD1 deubiqutinated and stabilized p53 through interaction via N-terminus of p53 and OTU domain of YOD1. In addition, expression levels of YOD1 were suppressed by elevated miR-221/222 in AML cells through binding to the 3' untranslated region of YOD1, as verified by reporter gene assays. Treatment of cells with miR-221/222 mimics and inhibitors yielded the expected effects on YOD1 expressions, in agreement with the negative correlation observed between the expression levels of miR-221/222 and YOD1 in AML cells. Finally, overexpression of YOD1 stabilized p53, upregulated pro-apoptotic p53 downstream genes, and increased the sensitivity of AML cells to FLT3 inhibitors remarkably. Collectively, our study identified a pathway connecting miR-221/222, YOD1, and p53 in AML. Targeting miR-221/222 and stimulating YOD1 activity may improve the therapeutic effects of FLT3 inhibitors in patients with AML.

PZR promotes tumorigenicity of lung cancer cells by regulating cell migration and invasion via modulating oxidative stress and cell adhesion.

PZR is a transmembrane glycoprotein encoded by the MPZL1 gene. It serves as a specific binding protein and substrate of tyrosine phosphatase SHP-2 whose mutations cause developmental diseases and cancers. Bioinformatic analyses of cancer gene databases revealed that PZR is overexpressed in lung cancer and correlated with unfavorable prognosis. To investigate the role of PZR in lung cancer, we employed the CRISPR technique to knockout its expression and recombinant lentiviruses to overexpress it in lung adenocarcinoma SPC-A1 cells. While knockout of PZR reduced colony formation, migration, and invasion, overexpression of PZR had the opposite effects. Furthermore, when implanted in immunodeficient mice, PZR-knockout SPC-A1 cells showed suppressed tumor-forming ability. Finally, the underlying molecular mechanism for these functions of PZR is its positive role in activating tyrosine kinases FAK and c-Src and in maintaining the intracellular level of reactive oxygen species (ROS). In conclusion, our data indicated that PZR plays an important role in lung cancer development, and it may serve as a therapeutic target for anti-cancer development and as a biomarker for cancer prognosis.

Sunitinib selectively targets leukemogenic signaling of mutant SHP2 in juvenile myelomonocytic leukemia.

Leukemogenic SHP2 mutations occur in 35% of patients with juvenile myelomonocytic leukemia (JMML), a hematopoietic malignancy with poor response to cytotoxic chemotherapy. Novel therapeutic strategies are urgently needed for patients with JMML. Previously, we established a novel cell model of JMML with HCD-57, a murine erythroleukemia cell line that depends on EPO for survival. SHP2-D61Y or -E76K drove the survival and proliferation of HCD-57 in absence of EPO. In this study, we identified sunitinib as a potent compound to inhibit SHP2-mutant cells by screening a kinase inhibitor library with our model. We used cell viability assay, colony formation assay, flow cytometry, immunoblotting, and a xenograft model to evaluate the effect of sunitinib against SHP2-mutant leukemia cells in vitro and in vivo. The treatment of sunitinib selectively induced apoptosis and cell cycle arrest in mutant SHP2-transformed HCD-57, but not parental cells. It also inhibited cell viability and colony formation of primary JMML cells with mutant SHP2, but not bone marrow mononuclear cells from healthy donors. Immunoblotting showed that the treatment of sunitinib blocked the aberrantly activated signals of mutant SHP2 with deceased phosphorylation levels of SHP2, ERK, and AKT. Furthermore, sunitinib effectively reduced tumor burdens of immune-deficient mice engrafted with mutant-SHP2 transformed HCD-57. Our data demonstrated that sunitinib selectively inhibited SHP2-mutant leukemia cells, which could serve as an effective therapeutic strategy for SHP2-mutant JMML in the future.

SHP2 participates in decidualization by activating ERK to maintain normal nuclear localization of progesterone receptor.

In brief: The establishment and maintenance of embryo implantation and pregnancy require decidualization of endometrial stromal cells. This paper reveals that SHP2 ensures the correct subcellular localization of progesterone receptor, thereby safeguarding the process of decidualization. Abstract: Decidualization is the process of conversion of endometrial stromal cells into decidual stromal cells, which is caused by progesterone production that begins during the luteal phase of the menstrual cycle and then increases throughout pregnancy dedicated to support embryonic development. Decidualization deficiency is closely associated with various pregnancy complications, such as recurrent miscarriage (RM). Here, we reported that Src-homology-2-containing phospho-tyrosine phosphatase (SHP2), a key regulator in the signal transduction process downstream of various receptors, plays an indispensable role in decidualization. SHP2 expression was upregulated during decidualization. SHP2 inhibitor RMC-4550 and shRNA-mediated SHP2 reduction resulted in a decreased level of phosphorylation of ERK and aberrant cytoplasmic localization of progesterone receptor (PR), coinciding with reduced expression of IGFBP1 and various other target genes of decidualization. Solely inhibiting ERK activity recapitulated these observations. Administration of RMC-4550 led to decidualization deficiency and embryo absorption in mice. Moreover, reduced expression of SHP2 was detected in the decidua of RM patients. Our results revealed that SHP2 is key to PR's nuclear localization, thereby indispensable for decidualization and that reduced expression of SHP2 might be engaged in the pathogenesis of RM.

Bioinformatics analyses of combined databases identify shared differentially expressed genes in cancer and autoimmune disease.

BACKGROUND: Inadequate immunity caused by poor immune surveillance leads to tumorigenesis, while excessive immunity due to breakdown of immune tolerance causes autoimmune genesis. Although the function of immunity during the onset of these two processes appears to be distinct, the underlying mechanism is shared. To date, gene expression data for large bodies of clinical samples are available, but the resemblances of tumorigenesis and autoimmune genesis in terms of immune responses remains to be summed up. METHODS: Considering the high disease prevalence, we chose invasive ductal carcinoma (IDC) and systemic lupus erythematosus (SLE) to study the potential commonalities of immune responses. We obtained gene expression data of IDC/SLE patients and normal controls from five IDC databases (GSE29044, GSE21422, GSE22840, GSE15852, and GSE9309) and five SLE databases (GSE154851, GSE99967, GSE61635, GSE50635, and GSE17755). We intended to identify genes differentially expressed in both IDC and SLE by using three bioinformatics tools including GEO2R, the limma R package, and Weighted Gene Co-expression Network Analysis (WGCNA) to perform function enrichment, protein-protein network, and signaling pathway analyses. RESULTS: The mRNA levels of signal transducer and activator of transcription 1 (STAT1), 2'-5'-oligoadenylate synthetase 1 (OAS1), 2'-5'-oligoadenylate synthetase like (OASL), and PML nuclear body scaffold (PML) were found to be differentially expressed in both IDC and SLE by using three different bioinformatics tools of GEO2R, the limma R package and WGCNA. From the combined databases in this study, the mRNA levels of STAT1 and OAS1 were increased in IDC while reduced in SLE. And the mRNA levels of OASL and PML were elevated in both IDC and SLE. Based on Kyoto Encyclopedia of Genes and Genomes pathway analysis and QIAGEN Ingenuity Pathway Analysis, both IDC and SLE were correlated with the changes of multiple components involved in the Interferon (IFN)-Janus kinase (JAK)-signal transducer and activator of transcription (STAT) signaling pathway. CONCLUSION: The expression levels of STAT1 and OAS1 manifest the opposite expression tendency across cancer and autoimmune disease. They are components in the IFN-JAK-STAT signaling pathway related to both tumorigenesis and autoimmune genesis. STAT1 and OAS1-associated IFN-JAK-STAT signaling could explain the commonalities during tumorigenesis and autoimmune genesis and render significant information for more precise treatment from the point of immune homeostasis.

Leukemogenic SHP2 mutations lead to erythropoietin independency of HCD-57 cells: a novel model for preclinical research of SHP2-mutant JMML.

Leukemogenic SHP2 mutations occur in 35% of patients with juvenile myelomonocytic leukemia (JMML), a rare but fatal hematopoietic malignancy without representative cell models, which are urgently needed to investigate the pathogenesis and to develop novel therapeutic strategies. In this study, we established stable cell lines with aberrant signaling resembling SHP2-mutant JMML through retroviral expression of SHP2-D61Y/E76K in HCD-57 cells, a murine erythroleukemia cell line that depends on erythropoietin (EPO) for survival. SHP2-D61Y/E76K drives the survival and proliferation of HCD-57 cells in the absence of EPO, but not in Ba/F3 cells in the absence of IL-3. Transformed HCD-57 cells showed activated MAPK signaling that is consistent with SHP2-mutant JMML. Transformed HCD-57 cells were sensitive to dasatinib and trametinib, two targeted drugs previously reported to inhibit SHP2-mutant JMML cells. Furthermore, we injected mutant SHP2-transformed HCD-57 cells into immune-deficient mice intravenously and found that these cells rapidly proliferated in the spleen and bone marrow, providing an excellent model for in vivo testing of drugs targeting the aberrant signaling of mutant SHP2. In conclusion, we established the novel cell lines HCD-57/SHP2-E76K and -D61Y that depended on signaling of mutant SHP2 for survival, thus resembling SHP2-mutant JMML. Our model is a valuable tool to investigate the pathogenic mechanisms of mutant SHP2 and targeted drugs for SHP2-mutant JMML.

Efficacy of SCF drug conjugate targeting c-KIT in gastrointestinal stromal tumor.

BACKGROUND: Gastrointestinal stromal tumor (GIST) is a rare type of cancer that occurs in the gastrointestinal tract. The majority of GIST cases carry oncogenic forms of KIT, the receptor for stem cell factor (SCF). Small molecule kinase inhibitor imatinib is effective in prolonging the survival of GIST patients by targeting KIT. However, drug resistance often develops during the therapeutic treatment. Here, we produced a SCF-emtansine drug conjugate (SCF-DM1) with favorable drug efficacy towards GIST cells. METHODS: Recombinant human SCF (rhSCF) was expressed in E. coli cells and further purified with Ni-NTA Sepharose and Phenyl Sepharose. It was then conjugated with DM1, and the conjugated product SCF-DM1 was evaluated using in vitro cell-based assays and in vivo xenograft mouse model. RESULTS: SCF-DM1 was effective in inhibiting imatinib-sensitive and -resistant GIST cell lines and primary tumor cells, with IC50 values of < 30 nM. It induced apoptosis and cell cycle arrest in GIST cells. In xenograft mouse model, SCF-DM1 showed favorable efficacy and safety profiles. CONCLUSIONS: rhSCF is a convenient and effective vector for drug delivery to KIT positive GIST cells. SCF-DM1 is an effective drug candidate to treat imatinib-sensitive and -resistant GIST.

HMGA1 chromatin regulators induce transcriptional networks involved in GATA2 and proliferation during MPN progression.

Myeloproliferative neoplasms (MPNs) transform to myelofibrosis (MF) and highly lethal acute myeloid leukemia (AML), although the actionable mechanisms driving progression remain elusive. Here, we elucidate the role of the high mobility group A1 (HMGA1) chromatin regulator as a novel driver of MPN progression. HMGA1 is upregulated in MPN, with highest levels after transformation to MF or AML. To define HMGA1 function, we disrupted gene expression via CRISPR/Cas9, short hairpin RNA, or genetic deletion in MPN models. HMGA1 depletion in JAK2V617F AML cell lines disrupts proliferation, clonogenicity, and leukemic engraftment. Surprisingly, loss of just a single Hmga1 allele prevents progression to MF in JAK2V617F mice, decreasing erythrocytosis, thrombocytosis, megakaryocyte hyperplasia, and expansion of stem and progenitors, while preventing splenomegaly and fibrosis within the spleen and BM. RNA-sequencing and chromatin immunoprecipitation sequencing revealed HMGA1 transcriptional networks and chromatin occupancy at genes that govern proliferation (E2F, G2M, mitotic spindle) and cell fate, including the GATA2 master regulatory gene. Silencing GATA2 recapitulates most phenotypes observed with HMGA1 depletion, whereas GATA2 re-expression partially rescues leukemogenesis. HMGA1 transactivates GATA2 through sequences near the developmental enhancer (+9.5), increasing chromatin accessibility and recruiting active histone marks. Further, HMGA1 transcriptional networks, including proliferation pathways and GATA2, are activated in human MF and MPN leukemic transformation. Importantly, HMGA1 depletion enhances responses to the JAK2 inhibitor, ruxolitinib, preventing MF and prolonging survival in murine models of JAK2V617F AML. These findings illuminate HMGA1 as a key epigenetic switch involved in MPN transformation and a promising therapeutic target to treat or prevent disease progression.

Bidirectional Interaction Between Cancer Cells and Platelets Provides Potential Strategies for Cancer Therapies.

Platelets are essential components in the tumor microenvironment. For decades, clinical data have demonstrated that cancer patients have a high risk of thrombosis that is associated with adverse prognosis and decreased survival, indicating the involvement of platelets in cancer progression. Increasing evidence confirms that cancer cells are able to induce production and activation of platelets. Once activated, platelets serve as allies of cancer cells in tumor growth and metastasis. They can protect circulating tumor cells (CTCs) against the immune system and detachment-induced apoptosis while facilitating angiogenesis and tumor cell adhesion and invasion. Therefore, antiplatelet agents and platelet-based therapies should be developed for cancer treatment. Here, we discuss the mechanisms underlying the bidirectional cancer-platelet crosstalk and platelet-based therapeutic approaches.

Tyrosine Kinase ROR1 as a Target for Anti-Cancer Therapies.

Receptor tyrosine kinase ROR1 plays an essential role in embryogenesis and is overexpressed in many types of malignant tumors. Studies have demonstrated that it plays an important role in oncogenesis by activating cell survival signaling events, particularly the non-canonical WNT signaling pathway. Antibody-based immunotherapies targeting ROR1 have been developed and evaluated in preclinical and clinical studies with promising outcomes. However, small molecule inhibitors targeting ROR1 are underappreciated because of the initial characterization of ROR1 as a peusdokinase. The function of ROR1 as a tyrosine kinase remains poorly understood, although accumulating evidence have demonstrated its intrinsic tyrosine kinase activity. In this review, we analyzed the structural and functional features of ROR1 and discussed therapeutic strategies targeting this kinase.

Expression of a recombinant FLT3 ligand and its emtansine conjugate as a therapeutic candidate against acute myeloid leukemia cells with FLT3 expression.

BACKGROUND: Most patients with acute myeloid leukemia (AML) remain uncurable and require novel therapeutic methods. Gain-of-function FMS-like tyrosine kinase 3 (FLT3) mutations are present in 30-40% of AML patients and serve as an attractive therapeutic target. In addition, FLT3 is aberrantly expressed on blasts in > 90% of patients with AML, making the FLT3 ligand-based drug conjugate a promising therapeutic strategy for the treatment of patients with AML. Here, E. coli was used as a host to express recombinant human FLT3 ligand (rhFL), which was used as a specific vehicle to deliver cytotoxic drugs to FLT3 + AML cells. METHODS: Recombinant hFL was expressed and purified from induced recombinant BL21 (DE3) E. coli. Purified rhFL and emtansine (DM1) were conjugated by an N-succinimidyl 3-(2-pyridyldithio)propionate (SPDP) linker. We evaluated the potency of the conjugation product FL-DM1 against FLT3-expressing AML cells by examining viability, apoptosis and the cell cycle. The activation of proteins related to the activation of FLT3 signaling and apoptosis pathways was detected by immunoblotting. The selectivity of FL-DM1 was assessed in our unique HCD-57 cell line, which was transformed with the FLT3 internal tandem duplication mutant (FLT3-ITD). RESULTS: Soluble rhFL was successfully expressed in the periplasm of recombinant E. coli. The purified rhFL was bioactive in stimulating FLT3 signaling in AML cells, and the drug conjugate FL-DM1 showed activity in cell signaling and internalization. FL-DM1 was effective in inhibiting the survival of FLT3-expressing THP-1 and MV-4-11 AML cells, with half maximal inhibitory concentration (IC50) of 12.9 nM and 1.1 nM. Additionally, FL-DM1 induced caspase-3-dependent apoptosis and arrested the cell cycle at the G2/M phase. Moreover, FL-DM1 selectively targeted HCD-57 cells transformed by FLT3-ITD but not parental HCD-57 cells without FLT3 expression. FL-DM1 can also induce obvious apoptosis in primary FLT3-positive AML cells ex vivo. CONCLUSIONS: Our data demonstrated that soluble rhFL can be produced in a bioactive form in the periplasm of recombinant E. coli. FL can be used as a specific vehicle to deliver DM1 into FLT3-expressing AML cells. FL-DM1 exhibited cytotoxicity in FLT3-expressing AML cell lines and primary AML cells. FL-DM1 may have potential clinical applications in treating patients with FLT3-positive AML.

Association of PTPN22-C1858T Polymorphism With Susceptibility to Mycobacterium tuberculosis and Mycobacterium leprae Infection: A Meta-Analysis.

It was previously published that single-nucleotide polymorphism rs2476601 (PTPN22 [protein tyrosine phosphatase non-receptor type 22]-C1858T) might be related to increased sensibility to Mycobacterium tuberculosis and M. leprae infection. However, the results were inconclusive despite a high degree of similarity between both parameters. Herein, we carried out this meta-analysis to systematically summarize and articulate the correlation between PTPN22-C1858T polymorphism and mycobacterial infection. The susceptibility of PTPN22-C1858T carriers with autoimmune conditions receiving immunosuppressive therapy to M. tuberculosis and M. leprae infection was determined. A systematic retrieval of studies on relevance of PTPN22-C1858T polymorphism to susceptibility of M. tuberculosis or M. leprae infection was performed in Chinese National Knowledge Infrastructure, PubMed and Embase databases. We regarded Odds ratios (ORs) and 95% confidence intervals (CIs) as the determined effect size. Finally, four and two case-control studies on tuberculosis and leprosy, respectively, were included. In all genetic models, without indicated association between PTPN22-C1858T polymorphism and tuberculosis's susceptibility. [C versus T: OR = 0.22 (95% CI: 0.09-0.50, PH = 0.887); CT versus CC: OR = 0.21 (95% CI: 0.09-0.49, PH = 0.889); TT+CT versus CC: OR = 0.21 (95% CI: 0.09-0.49, PH = 0.889)]. A significantly increased risk of leprosy was perceived in patients with the PTPN22-C1858T polymorphism [C versus T: OR = 2.82 (95% CI: 1.02-7.81, PH = 0.108)]. While the PTPN22-C1858T polymorphism is irrelevant to higher susceptibility to the infection of M. tuberculosis in Caucasians and Asians, it is relevant to increased susceptibility to the infection of M. leprae. However, the results of M. leprae are supposed to interpreted with prudence owing to the limited quantity of studies and heterogeneity. Further well-designed studies with sufficient populations are required to verify our conclusions.

Enhanced RIPK3 kinase activity-dependent lytic cell death in M1 but not M2 macrophages.

Macrophages play a crucial role in host innate immune defense against infection and tissue injury. Macrophages are highly plastic cells and their subtypes have been characterized as M1 (also termed classically activated) and M2 (alternatively activated). Although the M1/M2 paradigm has been well documented, less is known regarding the role of macrophage activation/polarization in inflammation-associated necrotic cell death. To address this gap in current knowledge, we prepared bone marrow-derived macrophages, induced them to M1 or M2 subtypes, and then investigated the expression of necroptosis signaling molecules and macrophage subtype-dependent responses to different necroptosis inducers. We found that necroptosis effector mixed lineage kinase domain-like protein (MLKL) and the key necroptosis regulator Z-DNA/RNA binding protein 1 were predominantly induced in M1 but not M2 macrophages. Interestingly, the protein but not mRNA levels of receptor-interacting protein kinase-3 (RIPK3) were also upregulated in M1 macrophages. We further found that macrophage necrotic cell death, the releases of lactate dehydrogenase and dead cell proteases as well as MLKL phosphorylation at Ser345 in response to various necroptosis inducers were greatly augmented in M1 but not M2 macrophages, and the accelerated effects were blocked by two structurally distinct specific RIPK3 inhibitors GSK872 or GSK843. Thus, our findings demonstrate that M1 but not M2 subtypes of macrophages are more susceptible to inflammation-related lytic cell death in an RIPK3 kinase activity-dependent manner.

Development of a highly sensitive method for detection of FLT3D835Y.

BACKGROUND: Acute myeloid leukemia (AML) is a malignant hematological neoplasm of myeloid progenitor cells. Mutations of FLT3 in its tyrosine kinase domain (FLT3-TKD) are found in ~ 8% of patients with AML, with D835Y as the most common substitution. This mutation activates survival signals that drives the disease and is resistant to the first generation FLT3 inhibitors. Development of a highly sensitive method to detect FLT3D835Y is important to direct therapeutic options, predict prognosis, and monitor minimal residual disease in patients with AML. METHODS AND RESULTS: In the present study, we developed a highly sensitive FLT3D835Y detection method by using the restriction fragment nested allele-specific PCR technique. The method consists of three steps: 1) initial amplification of DNA samples with PCR primers surrounding the FLT3D835Y mutation site, 2) digestion of the PCR products with restriction enzyme EcoRV that only cleaves the wild type allele, and 3) detection of FLT3D835Y by allele-specific PCR with nested primers. We were able to detect FLT3D835Y with a sensitivity of 0.001% by using purified plasmid DNAs and blood cell DNAs containing known proportions of FLT3D835Y. We analyzed blood cell DNA samples from 64 patients with AML and found six FLT3D835Y-positive cases, two of which could not be detected by conventional DNA sequencing methods. Importantly, the method was able to detect FLT3D835Y in a sample collected from a relapsed patient while the patient was in complete remission with negative MRD determined by flow cytometry. Therefore, our RFN-AS-PCR detected MRD after treatment that was missed by flow cytometry and Sanger DNA sequencing, by conventional methods. CONCLUSIONS: We have developed a simple and highly sensitive method that will allow for detection of FLT3D835Y at a very low level. This method may have major clinical implications for treatment of AML.

Proapoptotic Mitochondrial Carrier Homolog Protein PSAP Mediates Death Receptor 6 Induced Apoptosis.

Presenilin-associated protein (PSAP) was originally identified as a mitochondrial proapoptotic protein. To further explore the apoptotic pathway that involves PSAP, our yeast two-hybrid screen revealed that PSAP interacts with a death receptor, DR6. DR6 is a relatively less common member of the death receptor family and has been shown to mediate the neurotoxicity of amyloid-ß, mutant SOD1, and prion proteins and has also been implicated in the regulation of immune cell proliferation and differentiation. Our previous study showed that DR6 induces apoptosis via a unique mitochondria-dependent pathway different from the conventional death receptor-mediated extrinsic apoptotic pathways. Thus, the interaction of DR6 with PSAP established a direct molecular link between DR6 and mitochondrial apoptotic pathway. We investigated the possible role of PSAP in DR6-induced apoptosis. Interestingly, it was discovered that knockdown of PSAP strongly inhibited DR6-induced apoptosis. To further elucidate the mechanism by which PSAP mediates DR6-induced mitochondria-dependent apoptosis, our data demonstrated that knockdown of PSAP blocked DR6-induced Bax translocation and cytochrome c release from the mitochondria. Moreover, it was found that both PSAP and DR6 form complexes with Bax, but at different subcellular locations. The DR6-Bax complex was detected in the cytosolic fraction while the PSAP-Bax complex was detected in the mitochondrial fraction. The observation that knockdown of DR6 significantly reduced the amount of PSAP-Bax complex detected in mitochondria suggests a possibility that DR6-bound Bax is transferred to PSAP upon interaction with PSAP at the mitochondria, leading to cytochrome c release and eventually apoptosis.

Structure analysis of the receptor binding of 2019-nCoV.

2019-nCoV is a newly identified coronavirus with high similarity to SARS-CoV. We performed a structural analysis of the receptor binding domain (RBD) of spike glycoprotein responsible for entry of coronaviruses into host cells. The RBDs from the two viruses share 72% identity in amino acid sequences, and molecular simulation reveals highly similar ternary structures. However, 2019-nCoV has a distinct loop with flexible glycyl residues replacing rigid prolyl residues in SARS-CoV. Molecular modeling revealed that 2019-nCoV RBD has a stronger interaction with angiotensin converting enzyme 2 (ACE2). A unique phenylalanine F486 in the flexible loop likely plays a major role because its penetration into a deep hydrophobic pocket in ACE2. ACE2 is widely expressed with conserved primary structures throughout the animal kingdom from fish, amphibians, reptiles, birds, to mammals. Structural analysis suggests that ACE2 from these animals can potentially bind RBD of 2019-nCoV, making them all possible natural hosts for the virus. 2019-nCoV is thought to be transmitted through respiratory droplets. However, since ACE2 is predominantly expressed in intestines, testis, and kidney, fecal-oral and other routes of transmission are also possible. Finally, antibodies and small molecular inhibitors that can block the interaction of ACE2 with RBD should be developed to combat the virus.