Energetic determinants of the Par-3 interaction with the Par complex

The animal cell polarity regulator Par-3 recruits the Par complex (Par-6 and atypical Protein Kinase C–aPKC) to specific sites on the cell membrane. Although numerous physical interactions have been reported between Par-3 and the Par complex, it has been unclear how each contributes to the overall interaction. Using purified, intact Par complex and a quantitative binding assay, we found that energy for this interaction is provided by Par-3’s second and third PDZ protein interaction domains. Both Par-3 PDZ domains bind to aPKC’s PDZ Binding Motif (PBM) in the Par complex, with binding energy contributed from aPKC’s adjacent catalytic domain. In addition to highlighting the role of Par-3 PDZ interactions with the aPKC kinase domain and PBM in stabilizing Par-3 – Par complex assembly, our results indicate that each Par-3 molecule can potentially recruit two Par complexes to the membrane during cell polarization.

β-amyloid-driven synaptic depression requires PDZ protein interaction at AMPA-receptor subunit GluA3

Soluble oligomeric amyloid-β (Aβ) is a prime suspect to cause cognitive deficits in Alzheimer's disease and weakens synapses by removing AMPA-type glutamate receptors (AMPARs). We show that synapses of CA1 pyramidal neurons become vulnerable to Aβ when they express AMPAR subunit GluA3. We found that Aβ-oligomers reduce the levels of GluA3 immobilized at spines, indicating they deplete GluA3-containing AMPARs from synapses. These Aβ-driven effects critically depended on the PDZ-binding motif of GluA3. When GluA3 was expressed with a single amino acid mutation in its PDZ-binding motif that prevents GRIP binding, it did not end up at spines and Aβ failed to trigger synaptic depression. GluA3 with a different point mutation in the PDZ-motif that leaves GRIP-binding intact but prevents its endocytosis, was present at spines in normal amounts but was fully resistant to effects of Aβ. Our data indicate that Aβ-mediated synaptic depression requires the removal of GluA3 from synapses. We propose that GRIP-detachment from GluA3 is a critical early step in the cascade of events through which Aβ accumulation causes a loss of synapse.

A syntenin-deficient microenvironment educates AML for aggressiveness

In acute myeloid leukemia (AML), the stromal microenvironment plays a prominent role in promoting tumor cell survival and progression. Although widely explored, the crosstalk between leukemic and stromal cells remains poorly understood. Syntenin, a multi-domain PDZ protein, controls both the trafficking and signaling of key molecules involved in intercellular communication. Therefore, we aimed to clarify the role of environmental syntenin in the progression of AML. By in vivo approaches in syngeneic mice, we demonstrate that a syntenin-deficient environment reprograms AML blasts to survive independently of the stroma. Up-regulation of EEF1A2 in the blasts controls this gain of cell survival. Furthermore, using ex vivo co-culture systems, we show that syntenin-deficient bone marrow stromal cells (BMSC) enhance the survival of different types of AML cells, including patient samples, and suffice to educate syngeneic AML, recapitulating micro-environmental effects observed in vivo. We establish that syntenin-deficiency causes an increase of eIF5A and autophagy-related factors in BMSC, and provide evidence that the inhibition of autophagy prevents syntenin-deficient BMSC to stimulate AML survival. Altogether, these findings indicate that host-syntenin in the BM microenvironment acts as a repressor of AML aggressiveness.

Acquisition of a phospho-acceptor site enhances HPV E6 PDZ-binding motif functional promiscuity

All cancer-causing human papillomavirus (HPV) E6 oncoproteins have a C-terminal PDZ-binding motif (PBM), which correlates with oncogenic potential. Nonetheless, several HPVs with little or no oncogenic potential also have an E6 PBM, with minor sequence differences affecting PDZ protein selectivity. Furthermore, certain HPV types have a phospho-acceptor site embedded within the PBM. We therefore compared HPV-18, HPV-66 and HPV-40 E6 proteins to examine the possible link between the ability to target multiple PDZ proteins and the acquisition of a phospho-acceptor site. The mutation of essential residues in HPV-18E6 reduces its phosphorylation, and fewer PDZ substrates are bound. In contrast, the generation of consensus phospho-acceptor sites in HPV-66 and HPV-40 E6 PBMs increases the PDZ proteins recognized. Thus, although phosphorylation of the E6 PBM and PDZ protein recognition are mutually exclusive, they are closely linked, with the acquisition of a phospho-acceptor site also contributing to an expansion in the number of PDZ proteins bound.

Scribble co-operatively binds multiple α1D-adrenergic receptor C-terminal PDZ ligands

Many G protein-coupled receptors (GPCRs) are organized as dynamic macromolecular complexes in human cells. Unraveling the structural determinants of unique GPCR complexes may identify unique protein:protein interfaces to be exploited for drug development. We previously reported α1D-adrenergic receptors (α1D-ARs) – key regulators of cardiovascular and central nervous system function – form homodimeric, modular PDZ protein complexes with cell-type specificity. Towards mapping α1D-AR complex architecture, biolayer interferometry (BLI) revealed the α1D-AR C-terminal PDZ ligand selectively binds the PDZ protein scribble (SCRIB) with >8x higher affinity than known interactors syntrophin, CASK and DLG1. Complementary in situ and in vitro assays revealed SCRIB PDZ domains 1 and 4 to be high affinity α1D-AR PDZ ligand interaction sites. SNAP-GST pull-down assays demonstrate SCRIB binds multiple α1D-AR PDZ ligands via a co-operative mechanism. Structure-function analyses pinpoint R1110PDZ4 as a unique, critical residue dictating SCRIB:α1D-AR binding specificity. The crystal structure of SCRIB PDZ4 R1110G predicts spatial shifts in the SCRIB PDZ4 carboxylate binding loop dictate α1D-AR binding specificity. Thus, the findings herein identify SCRIB PDZ domains 1 and 4 as high affinity α1D-AR interaction sites, and potential drug targets to treat diseases associated with aberrant α1D-AR signaling.

Antagonistic Regulation of Circadian Output and Synaptic Development by the E3 Ubiquitin Ligase JETLAG and the DYSCHRONIC-SLOWPOKE Complex

SummaryCircadian output genes act downstream of the clock to promote rhythmic changes in behavior and physiology, yet their molecular and cellular functions are not well understood. Here we characterize an interaction between regulators of circadian entrainment, output and synaptic development in Drosophila that influences clock-driven anticipatory increases in morning and evening activity. We previously showed the JETLAG (JET) E3 Ubiquitin ligase resets the clock upon light exposure, while the PDZ protein DYSCHRONIC (DYSC) regulates circadian locomotor output and synaptic development. Surprisingly, we find that JET and DYSC antagonistically regulate synaptic development at the larval neuromuscular junction, and reduced JET activity rescues arrhythmicity of dysc mutants. Consistent with our prior finding that DYSC regulates SLOWPOKE (SLO) potassium channel expression, jet mutations also rescue circadian and synaptic phenotypes in slo mutants. Collectively, our data suggest that JET, DYSC and SLO promote circadian output in part by regulating synaptic morphology.HighlightsLoss of DYSC differentially impacts morning and evening oscillatorsReduced JET activity rescues the dysc and slo arrhythmic phenotypeReduced JET activity causes synaptic defects at the larval NMJJET opposes DYSC and SLO function at the NMJ synapse

Inhibitory effect of taspine derivative TAD1822-7 on tumor cell growth and angiogenesis via suppression of EphrinB2 and related signaling pathways

The aim of this study was to investigate the inhibitory effect of TAD1822-7, a synthesized taspine derivative, on cancer through its effects on tumor cell growth and angiogenesis via suppression of EphrinB2. The obtained data showed that TAD1822-7 decreased Bel-7402 cell viability and colony formation ability and suppressed cell migration. TAD1822-7 effectively inhibited blood vessel formation in an aortic ring assay to examine angiogenesis. Moreover, it also down regulated the expression of VEGFR2, VEGFR3, CD34, PLCγ, Akt, MMP2, MMP9, and CXCR4, and suppressed the expression of EphrinB2 and its PDZ protein, PICK1, in Bel-7402 cells. These results indicate that TAD1822-7 is a potential anti-angiogenic agent that can inhibit the viability and migration of Bel-7402 cells via suppression of EphrinB2 and the related signaling pathways.