A ubiquitin-proteasome pathway degrades the inner nuclear membrane protein Bqt4 to maintain nuclear membrane homeostasis.

Aberrant accumulation of inner nuclear membrane (INM) proteins is associated with deformed nuclear morphology and mammalian diseases. However, the mechanisms underlying the maintenance of INM homeostasis remain poorly understood. In this study, we explored the degradation mechanisms of the INM protein Bqt4 in the fission yeast Schizosaccharomyces pombe. We have previously shown that Bqt4 interacts with the transmembrane protein Bqt3 at the INM and is degraded in the absence of Bqt3. Here, we reveal that excess Bqt4, unassociated with Bqt3, is targeted for degradation by the ubiquitin-proteasome system localized in the nucleus and Bqt3 antagonizes this process. The degradation process involves the Doa10 E3 ligase complex at the INM. Bqt4 is a tail-anchored protein and the Cdc48 complex is required for its degradation. The C-terminal transmembrane domain of Bqt4 was necessary and sufficient for proteasome-dependent protein degradation. Accumulation of Bqt4 at the INM impaired cell viability with nuclear envelope deformation, suggesting that quantity control of Bqt4 plays an important role in nuclear membrane homeostasis.

A Guanidyl-Based Bivalent Peptidomimetic Inhibits K-Ras Prenylation and Association with c-Raf.

Unusual lipid modification of K-Ras makes Ras-directed cancer therapy a challenging task. Aiming to disrupt electrostatic-driven protein-protein interactions (PPIs) of K-Ras with FTase and GGTase I, a series of bivalent dual inhibitors that recognize the active pocket and the common acidic surface of FTase and GGTase I were designed. The structure-activity-relationship study resulted in 8 b, in which a biphenyl-based peptidomimetic FTI-277 was attached to a guanidyl-containing gallate moiety through an alkyl linker. Cell-based evaluation demonstrated that 8 b exhibited substantial inhibition of K-Ras processing without apparent interference with Rap-1A processing. Fluorescent imaging showed that 8 b disrupts localization of K-Ras to the plasma membrane and impairs interaction with c-Raf, whereas only FTI-277 was found to be inactive. These results suggest that targeting the PPI interface of K-Ras may provide an alternative method of inhibiting K-Ras.

Dipicolylamine/Metal Complexes that Promote Direct Cell-Membrane Penetration of Octaarginine.

Marked promotion of membrane permeation of a cell-penetrating peptide, octaarginine (R8), was attained by attachment to a single 2,2'-dipicolylamine moiety (DPA-R8) that forms 1:1 complexes with metal ions. Studies using giant unilamellar vesicles demonstrated that DPA targets phospholipids and enhances R8 binding to the membranes in the presence of metal ions. While DPA/Zn(II) complex has been most frequently employed for chelate formation with phosphates, Ni(II) had the most prominent effect on the membrane binding and penetration of DPA-R8. Facile cytosolic distribution of DPA-R8 was also attained in a few minutes in the presence of Ni(II). Analysis of the cellular uptake methods of DPA-R8/Ni(II) suggested the involvement of direct permeation through cell membrane without the use of endocytosis. The applicability of this system to the intracellular delivery of bioactive compounds was exemplified using a peptidomimetic farnesyltransferase inhibitor, FTI277.