Linker-extended native cyanovirin-N facilitates PEGylation and potently inhibits HIV-1 by targeting the glycan ligand

Cyanovirin-N (CVN) potently inhibits human immunodeficiency virus type 1 (HIV-1) infection, but both cytotoxicity and immunogenicity have hindered the translation of this protein into a viable therapeutic. A molecular docking analysis suggested that up to 12 residues were involved in the interaction of the reverse parallel CVN dimer with the oligosaccharide targets, among which Leu-1 was the most prominent hot spot residue. This finding provided a possible explanation for the lack of anti-HIV-1 activity observed with N-terminal PEGylated CVN. Therefore, linker-CVN (LCVN) was designed as a CVN derivative with a flexible and hydrophilic linker (Gly4Ser)3 at the N-terminus. The N-terminal α-amine of LCVN was PEGylated to create 10 K PEG-aldehyde (ALD)-LCVN. LCVN and 10 K PEG-ALD-LCVN retained the specificity and affinity of CVN for high mannose N-glycans. Moreover, LCVN exhibited significant anti-HIV-1 activity with attenuated cytotoxicity in the HaCaT keratinocyte cell line and MT-4 T lymphocyte cell lines. 10 K PEG-ALD-LCVN also efficiently inactivated HIV-1 with remarkably decreased cytotoxicity and pronounced cell-to-cell fusion inhibitory activity in vitro. The linker-extended CVN and the mono-PEGylated derivative were determined to be promising candidates for the development of an anti-HIV-1 agent. This derivatization approach provided a model for the PEGylation of biologic candidates without introducing point mutations. © 2014 Chen et al

IRTKS Promotes Insulin Signaling Transduction through Inhibiting SHIP2 Phosphatase Activity

Insulin signaling is mediated by a highly integrated network that controls glucose metabolism, protein synthesis, cell growth, and differentiation. Our previous work indicates that the insulin receptor tyrosine kinase substrate (IRTKS), also known as BAI1-associated protein 2-like 1 (BAIAP2L1), is a novel regulator of insulin network, but the mechanism has not been fully studied. In this work we reveal that IRTKS co-localizes with Src homology (SH2) containing inositol polyphosphate 5-phosphatase-2 (SHIP2), and the SH3 domain of IRTKS directly binds to SHIP2’s catalytic domain INPP5c. IRTKS suppresses SHIP2 phosphatase to convert phosphatidylinositol 3,4,5-triphosphate (PI(3,4,5)P3, PIP3) to phosphatidylinositol (3,4) bisphosphate (PI(3,4)P2). IRTKS-knockout significantly increases PI(3,4)P2 level and decreases cellular PI(3,4,5)P3 content. Interestingly, the interaction between IRTKS and SHIP2 is dynamically regulated by insulin, which feeds back and affects the tyrosine phosphorylation of IRTKS. Furthermore, IRTKS overexpression elevates PIP3, activates the AKT–mTOR signaling pathway, and increases cell proliferation. Thereby, IRTKS not only associates with insulin receptors to activate PI3K but also interacts with SHIP2 to suppress its activity, leading to PIP3 accumulation and the activation of the AKT–mTOR signaling pathway to modulate cell proliferation

Structural schematics of LCVN and the PEGylated product.

<p>The N-terminal glycine of LCVN and the serine-leucine joint between the linker and the CVN sequence are indicated. The blank rectangle represents the residual polypeptide of CVN. 10 KD mPEG-ALD was selectively reacted with the N-terminal α-amine of LCVN at different pKa values to create 10 K PEG-ALD-LCVN.</p

The anti-HIV-1 activity and MT-4 cytotoxicity of LCVNs (mean±SD, n = 3).

<p>IC₅₀, 50% viral inhibitory concentration;</p><p>CC₅₀, 50% cell inhibitory concentration;</p><p>SI, the ratio of CC₅₀ to IC₅₀.</p

Binding specificities of CVN to PA-oligosaccharides.

<p>The optimum (<b>A</b>) pH and (<b>B</b>) reaction time for CVN binding to PA-heptasaccharide (No. 19, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0086455#pone-0086455-g001" target="_blank">Figure 1</a>) were determined via centrifugal ultrafiltration-HPLC, and subsequently the (<b>C</b>) binding activity (specificity) of CVN and the 24 oligosaccharides was measured. The right panel in (<b>C</b>) depicts the non-reducing terminal Manα₁₋₂Man moieties in the D1, D2 or D3 arms of the Man₇₋₉GlcNAc₂ glycans that participated in the binding. Two independent experiments were performed for each PA-oligosaccharide, and the binding activity is presented as the average of the duplicate assays.</p