PDZ Domain-containing Protein Research Articles

Deciphering the alteration of MAP2 interactome caused by a schizophrenia-associated phosphorylation

Microtubule-associated protein 2 (MAP2) is a crucial regulator of dendritic structure and neuronal function, orchestrating diverse protein interactions within the microtubule network. We have shown MAP2 is hyperphosphorylated at serine 1782 (S1782) in schizophrenia and phosphomimetic mutation of S1782 in mice (MAP2S1782E) is sufficient to impair dendritic architecture. We sought to determine how this hyperphosphorylation affects the MAP2 interactome to provide insights into the disorder's mechanisms. We investigated the MAP2 interactome using co-immunoprecipitation and mass spectrometry in MAP2S1782E and MAP2WT mice. We found that S1782E MAP2 led to a substantial disruption of protein-protein interactions relative to WT MAP2. Reduced interactions with PDZ domain-containing proteins, calmodulin-binding proteins, ribosome proteins, and kinesin proteins may all contribute to dendritic impairments induced by S1782E, and may be linked to schizophrenia pathogenesis. Interestingly, novel gain-of-function interactions with PPM1L and KLHL8 nominated these as regulators of phosphoS1782 MAP2 abundance and potential therapeutic targets in schizophrenia.

HPV-18 E6 enhances the interaction between EMILIN2 and SNX27 to promote WNT signaling.

This study demonstrates that HPV E6 blocks EMILIN2 inhibition of WNT1 signaling, thereby enhancing cell proliferation in HPV-positive tumor cells. This involves a novel mechanism whereby the E6 PBM actually contributes toward enhancing the interaction between SNX27 and EMILIN2, suggesting that the mode of recognition of SNX27 by E6 and EMILIN2 is different. This is the first example of the E6 PBM altering a PDZ domain-containing protein to enhance potential substrate recognition.

A Potential Role for MAGI-1 in the Bi-Directional Relationship Between Major Depressive Disorder and Cardiovascular Disease.

Major Depressive Disorder (MDD) is characterized by persistent symptoms such as fatigue, loss of interest in activities, feelings of sadness and worthlessness. MDD often coexist with cardiovascular disease (CVD), yet the precise link between these conditions remains unclear. This review explores factors underlying the development of MDD and CVD, including genetic, epigenetic, platelet activation, inflammation, hypothalamic-pituitary-adrenal (HPA) axis activation, endothelial cell (EC) dysfunction, and blood-brain barrier (BBB) disruption. Single nucleotide polymorphisms (SNPs) in the membrane-associated guanylate kinase WW and PDZ domain-containing protein 1 (MAGI-1) are associated with neuroticism and psychiatric disorders including MDD. SNPs in MAGI-1 are also linked to chronic inflammatory disorders such as spontaneous glomerulosclerosis, celiac disease,ulcerative colitis, and Crohn's disease. Increased MAGI-1 expression has been observed in colonic epithelial samples from Crohn's disease and ulcerative colitis patients. MAGI-1 also plays a role in regulating EC activation and atherogenesis in mice and is essential for Influenza A virus (IAV) infection, endoplasmic reticulum stress-induced EC apoptosis, and thrombin-induced EC permeability. Despite being understudied in human disease; evidence suggests that MAGI-1 may play a role in linking CVD and MDD. Therefore, further investigation of MAG-1 could be warranted to elucidate its potential involvement in these conditions.

Modeling of host PDZ-dependent interactions with SARS-CoV-2 envelope protein and changes in PDZ protein expression in macrophages and dendritic cells.

PDZ (PSD-95 [postsynaptic density protein 95]/Dlg [Discs large]/ZO-1 [zonula occludens-1]) domain-containing proteins constitute a large family of scaffolds involved in a wide range of cellular tasks and are mainly studied in polarity functions. Diverse host PDZ proteins can be targeted by viral pathogens that express proteins containing PDZ-binding motifs (PDZbms). Previously, we have identified host PDZ-based interactions with the SARS-CoV-2 E protein (2E) in human monocytes. Here, we deepen the study of these interactions by docking and molecular dynamics analyses to identify the most favorable PDZ-PDZbm interaction of 7 host PDZ proteins with the PDZbm of 2E. In addition, we analyzed changes in the expression of 3 of the PDZ proteins identified as 2E interactors in monocytes (syntenin, ZO-2, and interleukin-16), in human monocyte-derived macrophages and in dendritic cells upon stimulation. Our results suggest that these PDZ proteins may have important functions in professional antigen-presenting cells, and their targeting by the PDZbm of 2E, a central virulence determinant of SARS-CoV-2, supports the hypothesis that such PDZ-dependent interaction in immune cells may constitute a viral evasion mechanism. An inhibitor design based on the PDZbm of 2E in the development of drugs against a variety of diseases is discussed.

SDCBP modulates tumor microenvironment, tumor progression and anti-PD1 efficacy in colorectal cancer.

Anti-programmed cell death 1 (aPD1) therapy has yielded limited success in patients with colorectal cancer (CRC). Syndecan binding protein (SDCBP), encodes a PDZ domain-containing protein that is essential for cellular processes, including cell adhesion, migration, and signal transduction. Here, we investigated the effect of SDCBP on tumor progression, immunotherapy, and the tumor microenvironment (TME) in CRC. High expression of SDCBP is associated with non-response to immunotherapy and correlated with poorer disease-free survival (DFS) in CRC patients. Inhibiting SDCBP by transfecting shRNA or using its inhibitor zinc pyrithione (ZnPT) hindered proliferation and metastasis while enhancing the efficacy of aPD1 treatment in a mouse xenograft model and liver metastasis model. The TME of CRC was significantly altered following ZnPT treatment characterized by a reduced amount of M2 macrophages and a heightened percentage of M1 macrophages. The co-culture system of CRC cells and macrophages provided evidence that SDCBP silencing promoted the repolarisation of M2 macrophages into M1. SDCBP promotes the proliferation, metastasis, and immunotherapy resistance of CRC. Thus, ZnPT represents an effective SDCBP inhibitor and exhibits considerable potential for combination with aPD1 to enhance immunotherapy efficacy.

Postnatal Pdzrn3 deficiency causes acute muscle atrophy without alterations in endplate morphology

PDZ domain-containing RING finger family protein 3 (PDZRN3) is expressed in various tissues, including the skeletal muscle. Although PDZRN3 plays a crucial role in the terminal differentiation of myoblasts and synaptic growth/maturation in myogenesis, the role of this molecule in postnatal muscles is completely unknown despite its lifelong expression in myofibers. In this study, we aimed to elucidate the function of PDZRN3 in mature myofibers using myofiber-specific conditional knockout mice. After tamoxifen injection, PDZRN3 deficiency was confirmed in both fast and slow myofibers of Myf6-CreERT2; Pdzrn3flox/flox (Pdzrn3mcKO) mice. Transcriptome analysis of the skeletal muscles of Pdzrn3mcKO mice identified differentially expressed genes, including muscle atrophy-related genes such as Smox, Amd1/2, and Mt1/2, suggesting that PDZRN3 is involved in the homeostatic maintenance of postnatal muscles. PDZRN3 deficiency caused muscle atrophy, predominantly in fast-twitch (type II) myofibers, and reduced muscle strength. While myofiber-specific PDZRN3 deficiency did not influence endplate morphology or expression of neuromuscular synaptic formation-related genes in postnatal muscles, indicating that the relationship between PDZRN3 and neuromuscular junctions might be limited during muscle development. Considering that the expression of Pdzrn3 in skeletal muscles was significantly lower in aged mice than in mature adult mice, we speculated that the PDZRN3-mediated muscle maintenance system might be associated with the pathophysiology of age-related muscle decline, such as sarcopenia.

A short sequence in the tail of SARS-CoV-2 envelope protein controls accessibility of its PDZ-binding motif to the cytoplasm

The carboxy-terminal tail of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) envelope protein (E) contains a PDZ-binding motif (PBM) which is crucial for coronavirus pathogenicity. During SARS-CoV-2 infection, the viral E protein is expressed within the Golgi apparatus membrane of host cells with its PBM facing the cytoplasm. In this work, we study the molecular mechanisms controlling the presentation of the PBM to host PDZ (PSD-95/Dlg/ZO-1) domain-containing proteins. We show that at the level of the Golgi apparatus, the PDZ-binding motif of the E protein is not detected by EC-terminal specific antibodies nor by the PDZ domain-containing protein-binding partner. Four alanine substitutions upstream of the PBM in the central region of the E protein tail is sufficient to generate immunodetection by anti-E antibodies and trigger robust recruitment of the PDZ domain-containing protein into the Golgi organelle. Overall, this work suggests that the presentation of the PBM to the cytoplasm is under conformational regulation mediated by the central region of the E protein tail and that PBM presentation probably does not occur at the surface of Golgi cisternae but likely at post-Golgi stages of the viral cycle.

PDZD2 Is Required for Normal Erythropoiesis and Its 37-KDa Secreted Product, sPDZD2, Functions As a Soluble Tumor Suppressor in AML

PDZ domain-containing protein 2 (PDZD2) is expressed in normal hematopoietic stem and progenitor cells (HSPC), specifically HSC, MPP, and erythroid progenitors, however, it is almost universally lost through hypermethylation in acute myeloid leukemia (AML). PDZD2 encodes for a 300-kDa protein containing 6 PDZ domains. The full-length protein is cleaved in the cytoplasm in a caspase 3-dependent manner, and the C-terminal fragment containing PDZ domains 5 and 6 is secreted to the tissue microenvironment as a 37-kDa protein known as secreted PDZD2 (sPDZD2). sPDZD2 has been reported to function as a tumor suppressor in some solid tumors, including breast, prostate, and liver cancer. Thus, we hypothesized that PDZD2 is required for normal hematopoiesis and that sPDZD2 functions as a soluble tumor suppressor in the hematopoietic system. We first sought to determine if sPDZD2 is secreted to the bone marrow (BM) microenvironment. Using an ELISA to quantify sPDZD2 levels in BM plasma isolated from healthy donors (n=6) and primary AMLs (n=20), we confirmed its secretion by normal CD34+ HSPCs (NBM) and downregulation in AMLs (NBM: 169 ± 17.53 vs AML: 46.12 ± 6.70 ng/ml; p<0.0001). In addition, AML cell lines (n=7) secreted significantly lower levels of sPDZD2 into the culture medium than normal HSPCs (n=2) (NBM: 28.71 ± 1.76 vs AML: 4.62 ± 1.19 ng/mL; p<0.0001). Next, to study the role of PDZD2 in normal hematopoiesis we performed in vitro myeloid and erythroid differentiation and colony-forming unit assays in CRISPR-Cas9-edited human HSPCs. CD34+ cells from healthy donors were electroporated with recombinant Cas9-sgRNA ribonucleoprotein complexes targeting PDZD2 and editing confirmed by T7I endonuclease assay and Sanger sequencing. PDZD2-edited cells (PDZD2 KO) were plated either on methylcellulose or liquid differentiation cultures (n=6). PDZD2 KO resulted in decreased colony number of all types (Control: 172.5 ± 34.37 vs PDZD2 KO: 137.0 ± 18.25, p<0.05), indicating reduced colony-forming potential. In addition, loss of PDZD2 resulted in a delay in terminal erythroid differentiation with an accumulation of pro-erythroblasts (CD71+CD235a lowCD105 high, p<0.05) and late basophilic erythroblasts (CD71+CD235a highCD105 int, p<0.01) as well as decreased orthochromatic erythroblasts (CD71+CD235a highCD105 neg, p<0.01) in a tri-phasic liquid culture system. By contrast, PDZD2 KO had no impact on myeloid differentiation. To determine the role of Pdzd2 in vivo, we took advantage of a Pdzd2 Gt/Gt gene-trap (GT) mouse model and performed peripheral blood (PB) and BM analysis compared to wild-type (WT) controls. PB counts at 2 months of age (n=6-9 per genotype) showed anemia, with a significant decrease in both red blood cell counts (WT: 9.04 ± 0.26 vs GT: 7.58 ± 0.47 x10 6/mL; p=0.026) and hemoglobin levels (WT: 14.88 ± 0.46 vs GT: 12.30 ± 0.81 g/dL; p=0.036), an observation compatible with the impaired erythropoiesis seen in vitro after PDZD2 KO in human HSPCs. In addition, we observed a trend to decrease megakaryocyte-erythroid progenitors (MEPs) and short-term HSCs in the BM of 3 months-old mice (n=3), though the small size of the cohort prevented robust statistical analysis. Next, we sought to evaluate the tumor suppressor role of sPDZD2 in the context of AML. We treated a panel of AML cell lines (n=9) and primary human AML specimens (n=4) with recombinant sPDZD2 ( r-sPDZD2; dose: 100nM-300nM/day) for a period of 8 days. AML cells showed dose-dependent growth inhibition (CellTiter-Glo, p<0.05 for all) and increased expression of either CD11b or CD15 differentiation markers. Cell cycle analysis on day 8 revealed that AML cells treated with r-sPDZD2 were arrested at G0/G1 phase compared to control. In summary, our study is the first to shed light on a previously unrecognized role for PDZD2 in hematopoiesis. Loss of PDZD2 in HSPC resulted in impaired erythroid differentiation both in vitro and in vivo. Moreover, our findings validate a soluble tumor suppressor role for sPDZD2 in AML, similar to that seen in solid tumors. Importantly, we further demonstrate a potential role for r-sPDZD2 as a novel therapeutic approach for AML.

TRIP6 disrupts tight junctions to promote metastasis and drug resistance and is a therapeutic target in colorectal cancer

Metastasis is the primary cause of death in colorectal cancer (CRC). Thyroid hormone receptor interacting protein 6 (TRIP6) is an adaptor protein that regulates cell motility. Here, we aim to elucidate the role of TRIP6 in driving CRC tumorigenesis and metastasis and evaluate its potential as a therapeutic target. TRIP6 mRNA is up-regulated in CRC compared to adjacent normal tissues in three independent cohorts (all P < 0.0001), especially in liver metastases (P < 0.001). High TRIP6 expression predicts poor prognosis of CRC patients in our cohort (P = 0.01) and TCGA cohort (P = 0.02). Colon-specific TRIP6 overexpression (Trip6KIVillin-Cre) in mice accelerated azoxymethane (AOM)-induced CRC (P < 0.05) and submucosal invasion (P < 0.0001). In contrast, TRIP6 knockout (Trip6+/− mice) slowed tumorigenesis (P < 0.05). Consistently, TRIP6 overexpression in CRC cells promoted epithelial-mesenchymal transition (EMT), cell migration/invasion in vitro, and metastases in vivo (all P < 0.05), whereas knockdown of TRIP6 exerted opposite phenotypes. Mechanistically, TRIP6 interacted PDZ domain-containing proteins such as PARD3 to impair tight junctions, evidenced by decreased tight junction markers and gut permeability dysfunction, inhibit PTEN, and activate oncogenic Akt signaling. TRIP6-induced pro-metastatic phenotypes and Akt activation depends on PARD3. Targeting TRIP6 by VNP-encapsulated TRIP6-siRNA synergized with Oxaliplatin and 5-Fluorouracil to suppress CRC liver metastases. In conclusion, TRIP6 promotes CRC metastasis by directly interacting with PARD3 to disrupt tight junctions and activating Akt signaling. Targeting of TRIP6 in combination with chemotherapy is a promising strategy for the treatment of metastatic CRC.

Suppression of the gene encoding PDZ domain-containing protein decreases cold tolerance and overwintering survival of the mosquito, Culex pipiens (Culicidae: Diptera).

In diapausing mosquitoes, cold tolerance and prolonged lifespan are important features that are crucial for overwintering success. In the mosquito Culex pipiens, we suggest that PDZ domain-containing protein (PDZ) (post synaptic density protein [PSD95], drosophila disc large tumor suppressor [Dlg1], and zonula occludens-1 protein [zo-1]) domain-containing protein is involved with these diapause features for overwintering survival in Culex mosquitoes. The expression level of pdz was significantly higher in diapausing adult females in the early stage in comparison to their nondiapausing counterparts. Suppression of the gene that encodes PDZ by RNA interference significantly decreased actin accumulation in the midgut of early-stage adult diapausing females. Inhibition of pdz also significantly reduced the survivability of diapausing females which indicates that this protein could play a key role in preserving the midgut tissues during early diapause.

Integrated metabolomics and phosphoproteomics reveal the protective role of exosomes from human umbilical cord mesenchymal stem cells in naturally aging mouse livers

BackgroundAging is characterized by a general decline in cellular function, which ultimately affects whole body homeostasis. This study aimed to investigate the effects and underlying mechanisms of exosomes derived from human umbilical cord mesenchymal stem cells (hUCMSC-exos) on the livers of naturally aging mice. MethodTwenty-two-month-old C57BL6 mice were used as a natural aging animal model, divided into a saline-treated wild‐type aged control group (WT-AC) and a hUCMSC-exo-treated group (WT-AEX), and then detected by morphology, metabolomics and phosphoproteomics. ResultsMorphological analysis showed that hUCMSC-exos ameliorated structural disorder and decreased markers of senescence and genome instability in aging livers. Metabolomics showed that hUCMSC-exos decreased the contents of saturated glycerophospholipids, palmitoyl-glycerols and eicosanoid derivatives associated with lipotoxicity and inflammation, consistent with the decreased phosphorylation of metabolic enzymes, such as propionate-CoA ligase (Acss2), at S267 detected by phosphoproteomics. Moreover, phosphoproteomics indicated that hUCMSC-exos reduced the phosphorylation of proteins participating in nuclear transport and cancer signaling, such as heat shock protein HSP90-beta (Hsp90ab1) at S226 and nucleoprotein TPR (Tpr) at S453 and S379, while increasing those involved in intracellular communication, such as calnexin (Canx) at S563 and PDZ domain-containing protein 8 (Pdzd8). Finally, phosphorylated HSP90β and Tpr were verified predominantly in hepatocytes. ConclusionHUCMSC-exos improved metabolic reprogramming and genome stability mainly associated with phosphorylated HSP90β in hepatocytes in natural aging livers. This work provides a comprehensive resource of biological data by omics to support future investigations of hUCMSC-exos in aging.

Additive energetic contributions of multiple peptide positions determine the relative promiscuity of viral and human sequences for PDZ domain targets.

Protein-protein interactions that involve recognition of short peptides are critical in cellular processes. Protein-peptide interaction surface areas are relatively small and shallow, and there are often overlapping specificities in families of peptide-binding domains. Therefore, dissecting selectivity determinants can be challenging. PDZ domains are a family of peptide-binding domains located in several intracellular signaling and trafficking pathways. These domains are also directly targeted by pathogens, and a hallmark of many oncogenic viral proteins is a PDZ-binding motif. However, amidst sequences that target PDZ domains, there is a wide spectrum in relative promiscuity. For example, the viral HPV16 E6 oncoprotein recognizes over double the number of PDZ domain-containing proteins as the cystic fibrosis transmembrane conductance regulator (CFTR) in the cell, despite similar PDZ targeting-sequences and identical motif residues. Here, we determine binding affinities for PDZ domains known to bind either HPV16 E6 alone or both CFTR and HPV16 E6, using peptides matching WT and hybrid sequences. We also use energy minimization to model PDZ-peptide complexes and use sequence analyses to investigate this difference. We find that while the majority of single mutations had marginal effects on overall affinity, the additive effect on the free energy of binding accurately describes the selectivity observed. Taken together, our results describe how complex and differing PDZ interactomes can be programmed in the cell.

Tax1 binding protein 3 regulates osteogenic and adipogenic differentiation through inactivating Wnt/β-catenin signalling.

Tax1 binding protein 3 (Tax1bp3) is a PDZ domain-containing protein that is overexpressed in cancer. Previous studies recognized Tax1bp3 as an inhibitor of β-catenin. Till now it is not known whether Tax1bp3 regulates osteogenic and adipogenic differentiation of mesenchymal progenitor cells. In the current study, the data showed that Tax1bp3 was expressed in bone and was increased in the progenitor cells when induced toward osteoblast and adipocyte differentiation. The overexpression of Tax1bp3 in the progenitor cells inhibited osteogenic differentiation and conversely stimulated adipogenic differentiation, and the knockdown of Tax1bp3 affected the differentiation of the progenitor cells oppositely. Ex vivo experiments using the primary calvarial osteoblasts from osteoblast-specific Tax1bp3 knock-in mice also demonstrated the anti-osteogenic and pro-adipogenic function of Tax1bp3. Mechanistic investigations revealed that Tax1bp3 inhibited the activation of canonical Wnt/β-catenin and bone morphogenetic proteins (BMPs)/Smads signalling pathways. Taken together, the current study has provided evidences demonstrating that Tax1bp3 inactivates Wnt/β-catenin and BMPs/Smads signalling pathways and reciprocally regulates osteogenic and adipogenic differentiation from mesenchymal progenitor cells. The inactivation of Wnt/β-catenin signalling may be involved in the reciprocal role of Tax1bp3.

Lipid-driven CFTR clustering is impaired in cystic fibrosis and restored by corrector drugs.

Some membrane proteins cluster in nanoscale cholesterol and sphingomyelin rich membrane domains, called lipid rafts, that transform into ceramide-rich rafts and coalesce into large ceramide-rich platforms during cell stress. However, the clustering mechanisms and their impact on membrane protein stability and function remain uncertain. We reported previously that the cystic fibrosis transmembrane conductance regulator (CFTR), which is mutated in the hyperinflammatory pulmonary disease cystic fibrosis (CF), forms clusters that are cholesterol-dependent and become incorporated into large and long-lived ceramide-rich platforms during hormonal stimulation. Using a combination of optimized fluorescence confocal imaging and quantitative analyses such as image correlation spectroscopy (ICS) and the k-space ICS (kICS), we report here that CFTR clustering at the plasma membrane of primary human bronchial epithelial cells does not involve known tethering interactions of CFTR with PDZ domain-containing proteins, filamin A or the actin cytoskeleton. It also does not require CFTR palmitoylation but is critically dependent on membrane lipid order and is induced by detergents that increase the phase separation of membrane lipids. Clustering and integration of CFTR into ceramide-rich platforms are impaired by the CF disease-causing mutations F508del and S13F and rescued by the CFTR modulators elexacaftor plus tezacaftor. These results indicate that CF therapeutics that correct mutant protein folding restore both CFTR trafficking and normal lipid interactions in the plasma membrane which results in its stability at the plasma membrane and its functional restoration.

PDZD2 Is Essential for Steady-State Hematopoiesis and Its 37-Kda Secreted Product, sPDZD2, Functions As a Soluble Tumor Suppressor in AML

PDZ domain-containing protein 2 (PDZD2) is expressed in normal hematopoietic stem and progenitor cells (HSPC), specifically HSC, MPP and erythroid progenitors, however, it is almost universally lost through hypermethylation in myeloid malignancies. PDZD2 encodes for a 300kDa protein containing 6 PDZ domains. The full-length protein is cleaved in the cytoplasm in a caspase 3-dependent manner, and the C-terminal fragment containing PDZ domains 5 and 6 is secreted to the tissue microenvironment as a 37kDa protein known as secreted PDZD2 (sPDZD2). Given that sPDZD2 has been reported to function as a tumor suppressor in solid tumors and its almost universal loss in AML, we hypothesized that PDZD2 is required for normal hematopoiesis and that sPDZD2 functions as a soluble tumor suppressor in the hematopoietic system. To determine if sPDZD2 is secreted to the bone marrow (BM) microenvironment we analyzed sPDZD2 levels in BM plasma isolated from primary AMLs and healthy donors using an ELISA-based detection method. We confirmed sPDZD2 secretion by normal CD34+ HSPC and detected significantly lower levels in AMLs (169 ± 17.53 vs 46.12 ± 6.70 ng/ml; p<0.0001). In addition, AML cell lines secreted significantly lower levels of sPDZD2 into the culture medium than HSPCs (28.71 ± 1.76 vs 4.62 ± 1.19 ng/mL; p<0.0001). Notably, treatment of a panel of AML cell lines (n=9) and primary human AML cells (n=4) with recombinant sPDZD2 (r-sPDZD2; dose: 100nM-300nM/day) led to growth inhibition (CellTiter-Glo, p<0.05 for all), differentiation (confirmed by upregulation of CD15/CD11b by flow cytometry) and cell cycle arrest in G0/G1. qRT-PCR analysis of cell cycle regulators in MV4-11 cells treated with r-sPDZD2 confirmed upregulation of CDKN2A (4.01-fold) and E2F4 (4.66-fold) and marked downregulation of multiple cyclin genes, including cyclins D and E, compatible with the cell cycle arrest observed by flow cytometry. To identify signaling pathways downstream of sPDZD2, we treated MV4-11 cells with 100nM r-sPDZD2 and performed phospho-kinase protein array followed by immunoblot for validation. In vitro treatment with r-sPDZD2 led to inhibition of ERK1/2 (T202/Y204, T185/Y187) and CREB (S133) phosphorylation, as well as decreased ß-Catenin expression and increased GSK3ß (S9), RSK1/2 (S221/S227) and p53 (S46) phosphorylation. In order to determine whether PDZD2 plays a role in normal hematopoiesis we performed in vitro differentiation in CRISPR-edited human HSPCs. We used a Cy3-labeled recombinant Cas9 protein and specific single guide RNAs (gRNAs) targeted against PDZD2. Cy3-positive cells were then used for in vitro myeloid or erythroid differentiation, and cell surface markers were evaluated by flow-cytometry analysis over a period of 11 days. While PDZD2-edited cells (PDZD2 KO) displayed a minor defect on myeloid differentiation due to insufficient upregulation of CD15, erythropoiesis appears to be more compromised, with a significant failure to adequately upregulate both CD235a (39 vs 15%) and CD71 (58 vs 26%). To determine the role of Pdzd2 in vivo, we took advantage of a Pdzd2Gt/Gt gene-trap knock-out mouse model and performed peripheral blood (PB) and BM analysis. PB counts at 8-weeks of age showed anemia, with a significant decrease in both red blood cell counts (9.04 ± 0.26 vs 7.58 ± 0.47 x106/µL) and hemoglobin levels (14.88 ± 0.46 vs 12.30 ± 0.81 g/dL) (p<0.05 for both), an observation compatible with the impaired erythropoiesis seen in vitro after PDZD2 KO in human HSPCs. In addition, we observed a trend to decreased MEPs and short-term HSCs, in the BM of 12-week-old mice (n=3), though the small size of the cohort prevented robust statistical analysis. In summary, our findings are the first to shed light on a previously unrecognized role for PDZD2 in hematopoiesis. Loss of PDZD2 in HSPC resulted in impaired erythroid differentiation both in vitro and in vivo. Moreover, our findings validate a soluble tumor suppressor role for sPDZD2 in AML, similar to that seen in solid tumors. Importantly, we further demonstrate a potential role for recombinant sPDZD2 as a novel therapeutic approach for AML.

Sunitinib and Pterostilbene Combination Treatment Exerts Antitumor Effects in Gastric Cancer via Suppression of PDZD8

The use of molecular-targeted drugs in the treatment of gastric cancer is increasing. However, the variety of molecular-targeted drugs in gastric cancer is still limited, and the development of new molecular-targeted therapies is required. The effect of combining sunitinib (SUN) with pterostilbene (PTE) on the human gastric cancer cell lines TMK1 and MKN74 was examined in in vitro and in vivo. Compared with SUN or PTE treatment alone, cotreatment induced pronounced suppression of cell proliferation, with a marked increase in oxidative stress. SUN was associated with a significant retention of mitochondrial Fe2+. SUN-treated cells decreased expression of PDZ domain-containing protein 8 (PDZD8). Knockdown of PDZD8 in both cells induced Fe2+ retention, and siPDZD8+PTE markedly suppressed cell proliferation with suppressed oxidative phosphorylation, as did the combination of SUN+PTE. In a nude mouse tumor model, a pronounced antitumor effect was observed with SUN+PTE treatment compared to SUN alone. PDZD8 may be a newly discovered off-target for SUN, and that the combined use of PTE with SUN significantly promotes antitumor activity in gastric cancer cell lines. The combined use of SUN and PTE might be a new molecular-targeted therapy for gastric cancer.

Circ_0020123 promotes cell proliferation and migration in lung adenocarcinoma via PDZD8.

High circ_0020123 expression is associated with poor prognosis in patients with non-small cell lung cancer (NSCLC) as previously reported. Whether circ_0020123 also plays an oncogenic role in lung adenocarcinoma (LUAD) is still unknown. Additionally, circ_0020123 is derived from part of exon (1312–3851) from its host gene PDZ domain-containing protein 8 (PDZD8). We hypothesized that circ_0020123 might affect malignant behaviors of LUAD cells by regulating PDZD8. Reverse transcription quantitative polymerase chain reaction revealed that PDZD8 was highly expressed in LUAD tissues and cells. PDZD8 knockdown suppressed LUAD cell proliferation and migration as shown by colony formation assays, Ethynyl deoxyuridine incorporation assays, Transwell assays, and wound healing assays. circ_0020123 was also found to be upregulated in LUAD tissues and cells. Moreover, circ_0020123 positively regulated PDZD8 expression in LUAD cells but exerted no significant effect on the transcriptional level of PDZD8. Mechanistically, circ_0020123 act as a competing endogenous RNA (ceRNA) to interact with miR-1283, thereby releasing the repression on PDZD8. Moreover, PDZD8 overexpression rescued the suppressive effect of circ_0020123 knockdown on LUAD cell proliferation and migration. In conclusion, circ_0020123 interacts with miR-1283 as a ceRNA to regulate PDZD8 expression, thus promoting the proliferation and migration of LUAD cells. The study might provide new biomarkers for future LUAD investigation.

Structural and biochemical analysis of the PTPN4 PDZ domain bound to the C-terminal tail of the human papillomavirus E6 oncoprotein.

High-risk genotypes of human papillomaviruses (HPVs) are directly implicated in various abnormalities associated with cellular hyperproliferation, including cervical cancer. E6 is one of two oncoproteins encoded in the HPV genome, which recruits diverse PSD-95/Dlg/ZO-1 (PDZ) domain-containing human proteins through its C-terminal PDZ-binding motif (PBM) to be degraded by means of the proteasome pathway. Among the three PDZ domain-containing protein tyrosine phosphatases, protein tyrosine phosphatase non-receptor type 3 (PTPN3) and PTPN13 were identified to be recognized by HPV E6 in a PBM-dependent manner. However, whether HPV E6 associates with PTPN4, which also has a PDZ domain and functions as an apoptosis regulator, remains undetermined. Herein, we present structural and biochemical evidence demonstrating the direct interaction between the PBM of HPV16 E6 and the PDZ domain of human PTPN4 for the first time. X-ray crystallographic structure determination and binding measurements using isothermal titration calorimetry demonstrated that hydrophobic interactions in which Leu158 of HPV16 E6 plays a key role and a network of intermolecular hydrogen bonds sustain the complex formation between PTPN4 PDZ and the PBM of HPV16 E6. In addition, it was verified that the corresponding motifs from several other high-risk HPV genotypes, including HPV18, HPV31, HPV33, and HPV45, bind to PTPN4 PDZ with comparable affinities, suggesting that PTPN4 is a common target of various pathogenic HPV genotypes.

SNX27 suppresses SARS-CoV-2 infection by inhibiting viral lysosome/late endosome entry

After binding to its cell surface receptor angiotensin converting enzyme 2 (ACE2), severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) enters the host cell through directly fusing with plasma membrane (cell surface pathway) or undergoing endocytosis traveling to lysosome/late endosome for membrane fusion (endocytic pathway). However, the endocytic entry regulation by host cell remains elusive. Recent studies show ACE2 possesses a type I PDZ binding motif (PBM) through which it could interact with a PDZ domain-containing protein such as sorting nexin 27 (SNX27). In this study, we determined the ACE2-PBM/SNX27-PDZ complex structure, and, through a series of functional analyses, we found SNX27 plays an important role in regulating the homeostasis of ACE2 receptor. More importantly, we demonstrated SNX27, together with retromer complex (the core component of the endosomal protein sorting machinery), prevents ACE2/virus complex from entering lysosome/late endosome, resulting in decreased viral entry in cells where the endocyticpathway dominates. The ACE2/virus retrieval mediated by SNX27-retromer could be considered as a countermeasure against invasion of ACE2 receptor-using SARS coronaviruses.

The Autocrine Secreted PDZD2 Functions As a Novel Tumor Suppressor in AML, Inducing Growth Inhibition and Cell Cycle Arrest

Epigenetic deregulation is a hallmark of acute myeloid leukemia (AML) and we have previously reported that recurring epigenetic abnormalities can be found across all AML subtypes, irrespective of cytogenetic or molecular drivers. Among the genes recurrently hypermethylated in AML we identified PDZD2 (PDZ domain-containing protein 2), which was silenced through DNA hypermethylation in over 90% of AML patients. PDZD2 processing generates two functional proteins: full-length PDZD2 and secreted PDZD2 (sPDZD2), a product of PDZD2 cleavage by caspase 3 at its C-terminal. While little is known about this protein's role in AML, work in solid tumors has shown that sPDZD2 can act as an autocrine tumor suppressor leading to senescence or quiescence of malignant cells. Given the role of PDZD2 in cancer and its almost universal loss in AML, we hypothesize that PDZD2 is a novel tumor suppressor in AML and that treatment with recombinant sPDZD2 represents a novel avenue for therapeutic targeting of AMLs.To test this hypothesis, we first characterized PDZD2 expression pattern in a panel of 13 AML cell lines and normal hematopoietic CD34 + cells. Quantification of PDZD2 mRNA levels in AML cell lines confirmed its almost universal downregulation when compared to normal CD34 + cells. Subcellular localization analysis in normal hematopoietic stem and progenitor cells demonstrated the presence of full-length PDZD2 in the cytoplasmic and nuclear fractions. In addition, treatment of a subset of the cell lines with the hypomethylating agent Decitabine at 500 nM for 72 h resulted in upregulation of PDZD2 by a range of 2.5 to 100-fold change, validating the observation that PDZD2 gets silenced through DNA hypermethylation. At the protein level, while full-length PDZD2 was detectable in approximately half the AML cell lines tested, detailed analysis revealed that in these cell lines PDZD2 was misprocessed, failing to get cleaved, and resulting in accumulation of the full-length version of the protein in the membranous (ER) fraction of the cell, while this is not observed in normal CD34 + cells.We next tested the effect of re-exposing AML cell lines to sPDZD2 by treating a panel of 10 AML cell lines with increasing doses of recombinant sPDZD2. sPDZD2 was added to the culture media every 12 h and cell growth was evaluated over a period of 8 days. We analyzed cell proliferation and viability every 48 h as well as cell cycle and expression of differentiation markers (CD11b and CD15) at the end of the treatment period. Even at relatively low doses (10-100nM), we observed variable degrees of dose-dependent growth inhibition in 8 out of 10 AML cell lines tested, with some cell lines showing greater and/or faster sensitivity than others. Cell cycle analysis showed that after 8 days of treatment, 5/8 sensitive cell lines (Kasumi1, KG1, SKNO1, MV4-11, HL60, K562) displayed G 0/G 1 arrest compared to vehicle control. Notably, 7/8 sensitive cell lines showed signs of a dose-dependent increase in the expression of either CD11b or CD15 differentiation markers, while the resistant cell line MOLM13 did not increase expression of either of these markers. In addition, patient-derived xenograft lines corresponding to AMLs carrying MLL-AF9 and 5 (out of 6 tested) primary human AML specimens cultured in vitro showed clear sensitivity to treatment with sPDZD2 compared to vehicle control. In contrast, normal CD34 + cells isolated from healthy donors and treated with sPDZD2 at the same doses showed no signs of toxicity, confirming sPDZD2's selectivity against AML cells. Finally, we sought to determine whether endogenous re-expression of full-length PDZD2 would likewise result in cell growth inhibition, indicating intact function of the cleavage mechanism. Transient activation of PDZD2 using a dCas9-VP64 fusion system resulted in a block in cell proliferation and G 0/G 1 cell cycle arrest in K562 cells, indicating that re-expression of PDZD2 phenocopies the results seen with sPDZD2 treatment.In summary, our findings are the first to demonstrate that sPDZD2 functions as a tumor suppressor in AML, inhibiting cell cycle and inducing differentiation, and that treatment with recombinant sPDZD2 can induce growth inhibition in a wide variety of AML cell lines and primary specimens, irrespective of their molecular and cytogenetic subtypes. DisclosuresNo relevant conflicts of interest to declare.