SPIN90, an adaptor protein, alters the proximity between Rab5 and Gapex5 and facilitates Rab5 activation during EGF endocytosis.

During ligand-mediated receptor endocytosis, the small GTPase Rab5 functions in vesicle fusion and trafficking. Rab5 activation is known to require interactions with its guanine nucleotide-exchange factors (GEFs); however, the mechanism regulating Rab5 interactions with GEFs remains unclear. Here, we show that the SH3-adapter protein SPIN90 participates in the activation of Rab5 through the recruitment of both Rab5 and its GEF, Gapex5, to endosomal membranes during epidermal growth factor (EGF)-mediated endocytosis. SPIN90 strongly interacts with the inactive Rab5/GDI2 complex through its C-terminus. In response to EGF signaling, extracellular signal-regulated kinase (ERK)-mediated phosphorylation of SPIN90 at Thr-242 enables SPIN90 to bind Gapex5 through its N-terminal SH3 domain. Gapex5 is a determinant of Rab5 membrane targeting, while SPIN90 mediates the interaction between Gapex5 and Rab5 in a phosphorylation-dependent manner. Collectively, our findings suggest that SPIN90, as an adaptor protein, simultaneously binds inactive Rab5 and Gapex5, thereby altering their spatial proximity and facilitating Rab5 activation.

Antibody targeting intracellular oncogenic Ras mutants exerts anti-tumour effects after systemic administration.

Oncogenic Ras mutants, frequently detected in human cancers, are high-priority anticancer drug targets. However, direct inhibition of oncogenic Ras mutants with small molecules has been extremely challenging. Here we report the development of a human IgG1 format antibody, RT11, which internalizes into the cytosol of living cells and selectively binds to the activated GTP-bound form of various oncogenic Ras mutants to block the interactions with effector proteins, thereby suppressing downstream signalling and exerting anti-proliferative effects in a variety of tumour cells harbouring oncogenic Ras mutants. When systemically administered, an RT11 variant with an additional tumour-associated integrin binding moiety for tumour tissue targeting significantly inhibits the in vivo growth of oncogenic Ras-mutated tumour xenografts in mice, but not wild-type Ras-harbouring tumours. Our results demonstrate the feasibility of developing therapeutic antibodies for direct targeting of cytosolic proteins that are inaccessible using current antibody technology.

Normalization of Tumor Vessels by Tie2 Activation and Ang2 Inhibition Enhances Drug Delivery and Produces a Favorable Tumor Microenvironment.

A destabilized tumor vasculature leads to limited drug delivery, hypoxia, detrimental tumor microenvironment, and even metastasis. We performed a side-by-side comparison of ABTAA (Ang2-Binding and Tie2-Activating Antibody) and ABA (Ang2-Blocking Antibody) in mice with orthotopically implanted glioma, with subcutaneously implanted Lewis lung carcinoma, and with spontaneous mammary cancer. We found that Tie2 activation induced tumor vascular normalization, leading to enhanced blood perfusion and chemotherapeutic drug delivery, markedly lessened lactate acidosis, and reduced tumor growth and metastasis. Moreover, ABTAA favorably altered the immune cell profile within tumors. Together, our findings establish that simultaneous Tie2 activation and Ang2 inhibition form a powerful therapeutic strategy to elicit a favorable tumor microenvironment and enhanced delivery of a chemotherapeutic agent into tumors.

Immunoglobulin Fc-fused, neuropilin-1-specific peptide shows efficient tumor tissue penetration and inhibits tumor growth via anti-angiogenesis.

Neuropilin-1 (NRP1) receptor, involved in vascular endothelial growth factor (VEGF)-mediated vascular permeability and tumor angiogenesis, is targeted by peptides that bind to its VEGF-binding site. However, these peptides also cross-react with the structurally related receptor, NRP2. Here, we describe an immunoglobulin Fc-fused peptide, Fc-TPP11, which specifically binds to the VEGF-binding site of NRP1 with approximately 2nM affinity, but negligibly to that of NRP2. Fc-TPP11 triggered NRP1-dependent signaling, enhanced vascular permeability via vascular endothelial (VE)-cadherin downregulation, and increased paracellular permeability via E-cadherin downregulation in tumor tissues. Fc-TPP11 also significantly enhanced the tumor penetration of co-injected anti-cancer drug, doxorubicin, leading to the improved in vivo anti-tumor efficacy. Fc-TPP11 was easily adapted to the full-length anti-epidermal growth factor receptor (EGFR) monoclonal antibody (mAb) cetuximab (Erbitux), cetuximab-TPP11, exhibiting more than 2-fold improved tumor penetration than the parent cetuximab. Fc-TPP11 exhibited a similar whole-body half-life to that of intact Fc in tumor bearing mice. In addition to the tumor-penetrating activity, Fc-TPP11 suppressed VEGF-dependent angiogenesis by blocking VEGF binding to NRP1, thereby inhibiting tumor growth without promoting metastasis in the mouse model. Our results show that NRP1-specific, high-affinity binding of Fc-TPP11, is useful to validate NRP1 signaling, independent of NRP2. Thus, Fc-TPP11 can be used as a tumor penetration-promoting agent with anti-angiogenic activity or directly adapted to mAb-TPP11 format for more potent anti-cancer antibody therapy.

A general strategy for generating intact, full-length IgG antibodies that penetrate into the cytosol of living cells.

Full-length IgG antibodies cannot cross cell membranes of living cells; this limits their use for direct targeting of cytosolic proteins. Here, we describe a general strategy for the generation of intact, full-length IgG antibodies, herein called cytotransmabs, which internalize into living cells and localize in the cytosol. We first generated a humanized light chain variable domain (VL) that could penetrate into the cytosol of living cells and was engineered for association with various subtypes of human heavy chain variable domains (VHs). When light chains with humanized VL were co-expressed with 3 heavy chains (HCs), including 2 HCs of the clinically approved adalimumab (Humira®) and bevacizumab (Avastin®), all 3 purified IgG antibodies were internalized into the cytoplasm of living cells. Cytotransmabs primarily internalized into living cells by the clathrin-mediated endocytic pathway through interactions with heparin sulfate proteoglycan that was expressed on the cell surface. The cytotransmabs escaped into the cytosol from early endosomes without being further transported into other cellular compartments, like the lysosomes, endoplasmic reticulum, Golgi apparatus, and nucleus. Furthermore, we generated a cytotransmab that co-localized with the targeted cytosolic protein when it was incubated with living cells, demonstrating that the cytotransmab can directly target cytosolic proteins. Internalized cytotransmabs did not show any noticeable cytotoxicity and remained in the cytosol for more than 6 h before being degraded by proteosomes. These results suggest that cytotransmabs, which efficiently enter living cells and reach the cytosolic space, will find widespread uses as research, diagnostic, and therapeutic agents.

F-actin-dependent regulation of NESH dynamics in rat hippocampal neurons.

Synaptic plasticity is an important feature of neurons essential for learning and memory. Postsynaptic organization and composition are dynamically remodeled in response to diverse synaptic inputs during synaptic plasticity. During this process, the dynamics and localization of postsynaptic proteins are also precisely regulated. NESH/Abi-3 is a member of the Abl interactor (Abi) protein family. Overexpression of NESH is associated with reduced cell motility and tumor metastasis. Strong evidence of a close relationship between NESH and the actin cytoskeleton has been documented. Although earlier studies have shown that NESH is prominently expressed in the brain, its function and characteristics are yet to be established. Data from the present investigation suggest that synaptic localization of NESH in hippocampal neurons is regulated in an F-actin-dependent manner. The dynamic fraction of NESH in the dendritic spine was analyzed using FRAP (fluorescence recovery after photobleaching). Interestingly, F-actin stabilization and disruption significantly affected the mobile fraction of NESH, possibly through altered interactions of NESH with the F-actin. In addition, NESH was synaptically targeted from the dendritic shaft to spine after induction of chemical LTP (long-term potentiation) and the translocation was dependent on F-actin. Our data collectively support the significance of the F-actin cytoskeleton in synaptic targeting of NESH as well as its dynamics.

NESH regulates dendritic spine morphology and synapse formation.

BACKGROUND: Dendritic spines are small membranous protrusions on the neuronal dendrites that receive synaptic input from axon terminals. Despite their importance for integrating the enormous information flow in the brain, the molecular mechanisms regulating spine morphogenesis are not well understood. NESH/Abi-3 is a member of the Abl interactor (Abi) protein family, and its overexpression is known to reduce cell motility and tumor metastasis. NESH is prominently expressed in the brain, but its function there remains unknown. METHODOLOGY/PRINCIPAL FINDINGS: NESH was strongly expressed in the hippocampus and moderately expressed in the cerebral cortex, cerebellum and striatum, where it co-localized with the postsynaptic proteins PSD95, SPIN90 and F-actin in dendritic spines. Overexpression of NESH reduced numbers of mushroom-type spines and synapse density but increased thin, filopodia-like spines and had no effect on spine density. siRNA knockdown of NESH also reduced mushroom spine numbers and inhibited synapse formation but it increased spine density. The N-terminal region of NESH co-sedimented with filamentous actin (F-actin), which is an essential component of dendritic spines, suggesting this interaction is important for the maturation of dendritic spines. CONCLUSIONS/SIGNIFICANCE: NESH is a novel F-actin binding protein that likely plays important roles in the regulation of dendritic spine morphogenesis and synapse formation.

P130Cas attenuates epidermal growth factor (EGF) receptor internalization by modulating EGF-triggered dynamin phosphorylation.

BACKGROUND: Endocytosis controls localization-specific signal transduction via epidermal growth factor receptor (EGFR), as well as downregulation of that receptor. Extracellular matrix (ECM)-integrin coupling induces formation of macromolecular complexes that include EGFR, integrin, Src kinase and p130Cas, resulting in EGFR activation. In addition, cell adhesion to ECM increases EGFR localization at the cell surface and reduces EGFR internalization. The molecular mechanisms involved are not yet well understood. METHODOLOGY/PRINCIPAL FINDINGS: We investigated the molecular mechanism by which p130Cas affects the endocytic regulation of EGFR. Biochemical quantification revealed that cell adhesion to fibronectin (FN) increases total EGFR levels and its phosphorylation, and that p130Cas is required for this process. Measurements of Texas Red-labeled EGF uptake and cell surface EGFR revealed that p130Cas overexpression reduces EGF-induced EGFR internalization, while p130Cas depletion enhances it. In addition, both FN-mediated cell adhesion and p130Cas overexpression reduce EGF-stimulated dynamin phosphorylation, which is necessary for EGF-induced EGFR internalization. Coimmunoprecipitation and GST pull-down assays confirmed the interaction between p130Cas and dynamin. Moreover, a SH3-domain-deleted form of p130Cas, which shows diminished binding to dynamin, inhibits dynamin phosphorylation and EGF uptake less effectively than wild-type p130Cas. CONCLUSIONS/SIGNIFICANCE: Our results show that p130Cas plays an inhibitory role in EGFR internalization via its interaction with dynamin. Given that the EGFR internalization process determines signaling density and specificity in the EGFR pathway, these findings suggest that the interaction between p130Cas and dynamin may regulate EGFR trafficking and signaling in the same manner as other endocytic regulatory proteins related to EGFR endocytosis.

The nuclear inclusion a (NIa) protease of turnip mosaic virus (TuMV) cleaves amyloid-ß.

BACKGROUND: The nuclear inclusion a (NIa) protease of turnip mosaic virus (TuMV) is responsible for the processing of the viral polyprotein into functional proteins. NIa was previously shown to possess a relatively strict substrate specificity with a preference for Val-Xaa-His-Gln↓, with the scissile bond located after Gln. The presence of the same consensus sequence, Val(12)-His-His-Gln(15), near the presumptive α-secretase cleavage site of the amyloid-ß (Aß) peptide led us to hypothesize that NIa could possess activity against Aß. METHODOLOGY/PRINCIPAL FINDINGS: Western blotting results showed that oligomeric as well as monomeric forms of Aß can be degraded by NIa in vitro. The specific cleavage of Aß was further confirmed by mass spectrometry analysis. NIa was shown to exist predominantly in the cytoplasm as observed by immunofluorescence microscopy. The overexpression of NIa in B103 neuroblastoma cells resulted in a significant reduction in cell death caused by both intracellularly generated and exogenously added Aß. Moreover, lentiviral-mediated expression of NIa in APP(sw)/PS1 transgenic mice significantly reduced the levels of Aß and plaques in the brain. CONCLUSIONS/SIGNIFICANCE: These results indicate that the degradation of Aß in the cytoplasm could be a novel strategy to control the levels of Aß, plaque formation, and the associated cell death.