Further progress towards understanding hepatic sinusoidal cells.

We have synthesized a series of MCM-41-type mesoporous silica nanoparticles (MSN). The surface of the MSNs are functionalized with 3-aminopropyl (AP), 3-guanidinopropyl (GP), 3-[N-(2-guanidinoethyl)guanidino]propyl (GEGP), and N-folate-3-aminopropyl (FAP). In contrast to the zeta-potential of -18.4 mV for FITC-MSN, the values of zeta-potential for AP-, GP-, GEGP-, and FAP-functionalized FITC-MSNs in 100 mM PBS buffer (pH 7.4) increased positively from -11.3, -10.6, -4.0, to +4.9 mV, respectively. The uptake efficiency, endocytosis mechanism, and biocompatibility of these organically functionalized MSNs were investigated with human cervical cancer cells (HeLa). Flow cytometry results suggested that the endocytosis of MSN could be manipulated by different surface functionalization. The immunocytochemistry study indicated that the uptake of these MSNs by HeLa cells was surface functional group dependent and involved several different mechanisms of endocytosis. Confocal fluorescence micrographs showed that the different surface functionalities of MSNs could also affect their ability to escape endosomal entrapment, which is a key factor in designing effective intracellular delivery vehicles.

Endocytosis↗

PubMed2006

Retrotranslocation of a viral A/B toxin from the yeast endoplasmic reticulum is independent of ubiquitination and ERAD.

K28 is a viral A/B toxin that traverses eukaryotic cells by endocytosis and retrograde transport through the secretory pathway. Here we show that toxin retrotranslocation from the endoplasmic reticulum (ER) requires Kar2p/BiP, Pdi1p, Scj1p, Jem1p, and proper maintenance of Ca(2+) homeostasis. Neither cytosolic chaperones nor Cdc48p/Ufd1p/Npl4p complex components or proteasome activity are required for ER exit, indicating that K28 retrotranslocation is mechanistically different from classical ER-associated protein degradation (ERAD). We demonstrate that K28 exits the ER in a heterodimeric but unfolded conformation and dissociates into its subunits as it emerges into the cytosol where beta is ubiquitinated and degraded. ER export and in vivo toxicity were not affected in a lysine-free K28 variant nor under conditions when ubiquitination and proteasome activity was blocked. In contrast, toxin uptake from the plasma membrane required Ubc4p (E2) and Rsp5p (E3) and intoxicated ubc4 and rsp5 mutants accumulate K28 at the cell surface incapable of toxin internalization. We propose a model in which ubiquitination is involved in the endocytic pathway of the toxin, while ER-to-cytosol retrotranslocation is independent of ubiquitination, ERAD and proteasome activity.

Endocytosis↗

PubMed2006

Endocytosis: the DUB version.

Dynamic modification of endosomal cargo proteins, such as the epidermal growth factor receptor, by ubiquitin can regulate their sorting into the lumen of multivesicular bodies through interactions with a complex protein network incorporating the endosomal sorting complexes required for transport (ESCRTs). Two deubiquitinating enzymes, AMSH and UBPY, interact with ESCRT protein components but exert opposite effects upon the rate of epidermal growth factor receptor downregulation. This might reflect their distinct specificities for different types of polyubiquitin chain linkage. We propose that AMSH might rescue ubiquitinated cargo from lysosomal degradation through disassembly of K63-linked polyubiquitin chains. UBPY function is essential for effective downregulation but is likely to be multifaceted, encompassing activity against both K63-linked and K48-linked polyubiquitin chains and including regulation of the stability of ESCRT-associated proteins such as STAM, by reversing their ubiquitination.

Endocytosis↗