Multiple PDZ domain scaffold protein Par-3 and phosphoinositides (PIPs) are required for polarity in diverse cell types. We show that the second PDZ domain of Par-3 binds to phosphatidylinositol (PI) lipid membranes with high affinity. We further demonstrate that a large subset of PDZ domains in mammalian genomes are capable of binding to PI lipid membranes, indicating that lipid binding is the second most prevalent interaction mode of PDZ domains known to date. The biochemical and structural basis of Par-3 PDZ2-mediated membrane interaction is characterized in detail. The membrane binding capacity of Par-3 PDZ2 is critical for epithelial cell polarization. Interestingly, the lipid phosphatase PTEN directly binds to the third PDZ domain of Par-3. The concatenation of the PIP-binding PDZ2 and the lipid phosphatase PTEN-binding PDZ3 endows Par-3 as an ideal scaffold protein for integrating PIP signaling events during cellular polarization.

Although cultural factors can pose a challenge to the application of PBL in non-Western settings, it appears that PBL can be applied in different cultural contexts. However, its globalisation does not postulate uniform processes and outcomes, and culturally sensitive alternatives might be developed.

Despite the availability of hospital-based screening and prenatal diagnosis for many years in Hong Kong, many women carrying fetuses at risk for thalassemia are not referred for genetic counseling. A community-based program of education, screening, and counseling is needed in Hong Kong and southern China.

The cytoplasmic domains of UNC5 are responsible for its netrin-mediated signaling events in axonal migrations, blood vessel patterning, and apoptosis, although the molecular mechanisms governing these processes are unknown. To provide a foundation for the elucidation of the UNC5-mediated signaling mechanism, we determined the crystal structure of the cytoplasmic portion of UNC5b. We found that it contains three distinctly folded domains, namely ZU5, UPA, and death domain (DD). These three domains form a structural supramodule, with ZU5 binding to both UPA and DD, thereby locking the ZU5-UPA-DD supramodule in a closed conformation and suppressing its biological activities. Release of the closed conformation of the ZU5-UPA-DD supramodule leads to the activation of the receptor in the promotion of apoptosis and blood vessel patterning. Finally, we provide evidence showing that the supramodular nature of UNC5 ZU5-UPA-DD is likely to be shared by the ankyrin and PIDD families of scaffold proteins.

PDZ domain-containing scaffold protein Par-3 is the central organizer of the evolutionarily conserved cell polarity-regulatory Par-3⅐Par-6⅐atypical protein kinase C complex. The PDZ domains of Par-3 have also been implicated as potential phosphoinositide signaling integrators, since its second PDZ domain binds to phosphoinositides, and the third PDZ interacts with phosphoinositide phosphatase PTEN. However, the molecular basis of Par-3/PTEN interaction is still poorly understood. Additionally, it is not known whether the regulatory function of PTEN in cell polarity is specifically mediated by its interaction with Par-3. The structures of Par-3 PDZ3 in both its free and PTEN tail peptide-bound forms determined in this work reveal that Par-3 PDZ3 binds to PTEN with two discrete binding sites: a canonical PDZ-ligand interaction site and a distal, opposite charge-charge interaction site. This distinct target recognition mechanism confers the interaction specificity of the Par-3⅐PTEN complex. We show that the Par-3 PDZ3-PTEN binding is required for the enrichment of PTEN at the junctional membranes of Madin-Darby canine kidney cells. Finally, we demonstrate that the junctional membrane-localized PTEN is specifically required for the polarization of Madin-Darby canine kidney cells. These results, together with earlier data, firmly establish that Par-3 functions as a scaffold in integrating phosphoinositide signaling events during cellular polarization.The polarization of cells is a fundamental process required for the differentiation, proliferation, and morphogenesis of both unicellular and multicellular organisms (1-5). Several common sets of proteins, initially identified through genetic screenings, are known to be essential for the establishment and maintenance of cell polarity (for reviews, see Refs. 6 -11). Par (partitioning-defective) proteins, originally discovered in studies of embryonic asymmetric cell division in the Caenorhabditis elegans zygote, are one set of these obligatory cell polarity-controlling proteins (7,(12)(13)(14)(15). Among the Par proteins, Par-3 and Par-6 are PDZ domain-containing scaffold proteins, and they often localize and function together in places such as the anterior of the C. elegans zygote during asymmetric embryonic cell division (16,17). Both Par-3 and Par-6 can directly interact with atypical protein kinase C (aPKC).3 This evolutionarily conserved Par-3⅐Par-6⅐aPKC complex is absolutely required for the establishment and maintenance of the polarity of essentially all multicellular eukaryotic cells (18 -20).Par-3 contains an N-terminal domain followed by three PDZ domains and an extended C terminus. In addition to binding directly to both Par-6 and aPKC, the N-terminal domain of Par-3 can self-oligomerize, thereby promoting the formation of large assemblies of the Par-3⅐Par-6⅐aPKC macromolecular complexes (18,19,21). Thus, Par-3 is viewed as the central organizer of the Par-3⅐Par-6⅐aPKC assembly. Among the three PDZ domains of Par-3, the first PDZ domain was reported to bind to a...

Epigenetic therapies are emerging strategies to prime host immune system to immunotherapies. Here we show that depletion of H3K4 demethylase KDM5B in melanoma cells induces anti-tumor T cell immunity through up-regulation of retroelements, which activates cytosolic nucleic acid sensing pathways and subsequent type I interferon responses. Mechanistically, KDM5B recruits H3K9 methyltransferase SETDB1 to the chromatin to repress these retroelements. Ablation of KDM5B enhances responses of poorly immunogenic melanoma tumors to anti PD-1 treatment. Our studies suggest that therapies targeting KDM5B are a novel approach to enhance tumor immunogenicity and overcome immunotherapy resistance. MainKDM5B expression is negatively correlated with anti-tumor immunity and response to immune checkpoint blockade. To explore the roles of KDM5B in melanoma, we performed Gorilla Gene Ontology analysis of 9,336 genes negatively correlated with KDM5B expression in TCGA melanoma dataset. The top 5 gene ontology categories anti-correlated with KDM5B expression are all related to immune system processes (Fig. 1a). To further examine the relationship of KDM5B with anti-tumor immune responses, we correlated KDM5B mRNA levels with T cell markers, antigen presentation and cytokine genes in TCGA melanoma dataset. Remarkably, KDM5B expression is inversely associated with all the genes examined, including IFNG (IFN-γ) and TNF (TNF-α), two crucial effector cytokines in anti-tumor immunity 1 (Extended Data Table 1). These results suggest that KDM5B expression is inversely correlated with intra-tumoral in ammation. Consistently, patients with higher KDM5B expression have lower expression of markers for CD8 + T cells in the tumors in TCGA melanoma dataset (Extended Data Fig. 1a). Lack of T cell in ltration in pre-treatment biopsies normally correlates with resistance to immune checkpoint blockade (ICB) 2 . To evaluate whether KDM5B levels predict response to immune checkpoint blockade in melanoma, we analyzed RNA-seq data of pre-treatment specimens from tumors sampled prior to anti-PD-1 therapy 3 . We found that KDM5B expression levels are signi cantly lower in patients with complete response than those in patients with progressive disease (Fig. 1b and Extended Data Table 2). Furthermore, we evaluated KDM5B protein levels in a tissue microarray (TMA) containing samples from patients treated with ICB.Patients who responded to ICB had a very low intensity of KDM5B staining and lacked detectable KDM5B high melanoma cells, while the non-responders had a major subset of KDM5B high melanoma cells (Extended Data Fig. 1b). These data raised the possibility that KDM5B could be a biomarker for poor response to ICB and a KDM5B targeting therapy might overcome resistance to ICB. KDM5B loss induces anti-tumor immunity in a murine model of melanomaTo test whether ablation of KDM5B in melanoma induces anti-tumor immunity, we assessed the effects of Kdm5b deletion in the immunogenic YUMMER1.7 mouse melanoma cells 4 on their tumor growth in syngeneic C57BL/6J mice ...

The tumor suppressor protein p53 is known to undergo cytoplasmic dynein-dependent nuclear translocation in response to DNA damage. However, the molecular link between p53 and the minus end-directed microtubule motor dynein complex has not been described. We report here that the 8-kDa light chain (LC8) of dynein binds to p53-binding protein 1 (53BP1). The LC8-binding domain was mapped to a short peptide segment immediately N-terminal to the kinetochore localization region of 53BP1. The LC8-binding domain is completely separated from the p53-binding domain in 53BP1. Therefore, 53BP1 can potentially act as an adaptor to assemble p53 to the dynein complex. Unlike other known LC8-binding proteins, 53BP1 contains two distinct LC8-binding motifs that are arranged in tandem. We further showed that 53BP1 can directly associate with the dynein complex. Disruption of the interaction between LC8 and 53BP1 in vivo prevented DNA damageinduced nuclear accumulation of p53. These data illustrate that LC8 is able to function as a versatile acceptor to link a wide spectrum of molecular cargoes to the dynein motor.

Minocycline has been shown to have remarkably neuroprotective qualities, but underlying mechanisms remain elusive. We reported here the robust neuroprotection by minocycline against glutamate-induced apoptosis through regulations of p38 and Akt pathways. Pre-treatment of cerebellar granule neurons (CGNs) with minocycline (10-100 lM) elicited a dose-dependent reduction of glutamate excitotoxicity and blocked glutamate-induced nuclear condensation and DNA fragmentations. Using patch-clamping and fluorescence Ca 2+ imaging techniques, it was found that minocycline neither blocked NMDA receptors, nor reduced glutamatecaused rises in intracellular Ca 2+. Instead, confirmed by immunoblots, minocycline in vivo and in vitro was shown to directly inhibit the activation of p38 caused by glutamate. A p38-specific inhibitor, SB203580, also attenuated glutamate excitotoxicity. Furthermore, the neuroprotective effects of minocycline were blocked by phosphatidylinositol 3-kinase (PI3-K) inhibitors LY294002 and wortmannin, while pharmacologic inhibition of glycogen synthase kinase 3b (GSK3b) attenuated glutamate-induced apoptosis. In addition, immunoblots revealed that minocycline reversed the suppression of phosphorylated Akt and GSK3b caused by glutamate, as were abolished by PI3-K inhibitors. These results demonstrate that minocycline prevents glutamate-induced apoptosis in CGNs by directly inhibiting p38 activity and maintaining the activation of PI3-K/Akt pathway, which offers a novel modality as to how the drug exerts protective effects.