Both the sec6/8 complex and septin filaments have been implicated in directing vesicles and proteins to sites of active membrane addition in yeast. The rat brain sec6/8 complex coimmunoprecipitates with a filament composed of four mammalian septins, suggesting an interaction between these complexes. One of the septins, CDC10, displays broad subcellular and tissue distributions and is found in postmitotic neurons as well as dividing cells. Electron microscopic studies showed that the purified rat brain septins form filaments of 8.25 nm in diameter; the lengths of the filaments are multiples of 25 nm. Glutaraldehyde-fixed rat brain sec6/8 complex adopts a conformation resembling the letter "T" or "Y". The sec6/8 and septin complexes likely play an important role in trafficking vesicles and organizing proteins at the plasma membrane of neurons.

The degree of stability of antibody-drug linkers in systemic circulation, and the rate of their intracellular processing within target cancer cells are among the key factors determining the efficacy of antibody-drug conjugates (ADC) in vivo. Previous studies demonstrated the susceptibility of cleavable linkers, as well as auristatin-based payloads, to enzymatic cleavage in rodent plasma. Here, we identify Carboxylesterase 1C as the enzyme responsible for the extracellular hydrolysis of valine-citrulline-paminocarbamate (VC-PABC)-based linkers in mouse plasma. We further show that the activity of Carboxylesterase 1C towards VC-PABC-based linkers, and consequently the stability of ADCs in mouse plasma, can be effectively modulated by small chemical modifications to the linker. While the introduced modifications can protect the VC-PABC-based linkers from extracellular cleavage, they do not significantly alter the intracellular linker processing by the lysosomal protease Cathepsin B. The distinct substrate preference of the serum Carboxylesterase 1C offers the opportunity to modulate the extracellular stability of cleavable ADCs without diminishing the intracellular payload release required for ADC efficacy. Mol Cancer Ther; 15(5); 958-70. Ó2016 AACR.

The molecules that specify domains on the neuronal plasma membrane for the delivery and accumulation of vesicles during neurite outgrowth and synapse formation are unknown. We investigated the role of the sec6/8 complex, a set of proteins that specifies vesicle targeting sites in yeast and epithelial cells, in neuronal membrane trafficking. This complex was found in layers of developing rat brain undergoing synaptogenesis. In cultured hippocampal neurons, the sec6/8 complex was present in regions of ongoing membrane addition: the tips of growing neurites, filopodia, and growth cones. In young axons, the sec6/8 complex was also confined to periodic domains of the plasma membrane. The distribution of synaptotagmin, synapsin1, sec6, and FM1-43 labeling in cultured neurons suggested that the plasma membrane localization of the sec6/8 complex preceded the arrival of synaptic markers and was downregulated in mature synapses. We propose that the sec6/8 complex specifies sites for targeting vesicles at domains of neurite outgrowth and potential active zones during synaptogenesis.

The trans-Golgi network (TGN) plays a pivotal role in directing proteins in the secretory pathway to the appropriate cellular destination. VAMP4, a recently discovered member of the vesicle-associated membrane protein (VAMP) family of trafficking proteins, has been suggested to play a role in mediating TGN trafficking. To better understand the function of VAMP4, we examined its precise subcellular distribution. Indirect immunofluorescence and electron microscopy revealed that the majority of VAMP4 localized to tubular and vesicular membranes of the TGN, which were in part coated with clathrin. In these compartments, VAMP4 was found to colocalize with the putative TGN-trafficking protein syntaxin 6. Additional labeling was also present on clathrin-coated and noncoated vesicles, on endosomes and the medial and trans side of the Golgi complex, as well as on immature secretory granules in PC12 cells. Immunoprecipitation of VAMP4 from rat brain detergent extracts revealed that VAMP4 exists in a complex containing syntaxin 6. Converging lines of evidence implicate a role for VAMP4 in TGN-to-endosome transport. INTRODUCTIONSecretory pathway compartments can be subdivided into two central membrane populations, the endoplasmic reticulum (ER)-Golgi system and the trans-Golgi network (TGN)-endosomal system. The ER-Golgi system performs the folding, oligomerization, and coand post-translational modifications of proteins transiting the secretory pathway. The TGN-endosomal system is central to the sorting, export, and recycling of numerous soluble and membrane-associated lysosomal and secretory pathway proteins. At the TGN, newly synthesized proteins are routed to endosomes and lysosomes, to regulated and constitutive exocytic pathways, and, in polarized cells, to apical and basolateral membranes (Palade, 1975;Mellman and Simons, 1992;Rothman and Wieland, 1996;Schekman and Orci, 1996;Traub and Kornfeld, 1997). The TGN also receives membrane traffic from the endocytic pathway; e.g., the two mannose 6-phosphate receptors (MPRs) carry lysosomal hydrolases from the TGN to late endosomes, where they release the hydrolases and then return to the TGN for another round of transport (Kornfeld, 1992). Movement of protein between these compartments occurs by the budding and fusion of transport vesicles. Maintaining the identity of membrane-bound compartments in the face of the massive flux between them requires the orchestrated interaction of a large number of components, including lipid, cytosolic proteins such as ATPases and GTPases and integral membrane proteins present on target membranes and transport vesicles.The most intensely studied vesicle and target membrane proteins are members of the vesicle-associated membrane protein (VAMP)/synaptobrevin, syntaxin, and synaptosomal-associated protein of 25 kDa (SNAP-25) families. These proteins, also known as soluble N-ethylmaleimide-sensitive factor (NSF) at- ‡ Corresponding author. E-mail address: scheller@cmgm. stanford.edu.© 1999 by The American Society for Cell Biology 1957 tachm...

No abstract

The systemic stability of the antibody−drug linker is crucial for delivery of an intact antibody−drug conjugate (ADC) to target-expressing tumors. Linkers stable in circulation but readily processed in the target cell are necessary for both safety and potency of the delivered conjugate. Here, we report a range of stabilities for an auristatin-based payload site-specifically attached through a cleavable valine-citrulline-p-aminobenzylcarbamate (VC-PABC) linker across various sites on an antibody. We demonstrate that the conjugation site plays an important role in determining VC-PABC linker stability in mouse plasma, and that the stability of the linker positively correlates with ADC cytotoxic potency both in vitro and in vivo. Furthermore, we show that the VC-PABC cleavage in mouse plasma is not mediated by Cathepsin B, the protease thought to be primarily responsible for linker processing in the lysosomal degradation pathway. Although the VC-PABC cleavage is not detected in primate plasma in vitro, linker stabilization in the mouse is an essential prerequisite for designing successful efficacy and safety studies in rodents during preclinical stages of ADC programs. The divergence of linker metabolism in mouse plasma and its intracellular cleavage offers an opportunity for linker optimization in the circulation without compromising its efficient payload release in the target cell.

No abstract

No abstract