Recombinant v- and t-SNARE proteins reconstituted into separate lipid bilayer vesicles assemble into SNAREpins-SNARE complexes linking two membranes. This leads to spontaneous fusion of the docked membranes at physiological temperature. Docked unfused intermediates can accumulate at lower temperatures and can fuse when brought to physiological temperature. A supply of unassembled v- and t-SNAREs is needed for these intermediates to form, but not for the fusion that follows. These data imply that SNAREpins are the minimal machinery for cellular membrane fusion.

Glutamine serves as an important source of energy and building blocks for many tumor cells. The first step in glutamine utilization is its conversion to glutamate by the mitochondrial enzyme glutaminase. CB-839 is a potent, selective, and orally bioavailable inhibitor of both splice variants of glutaminase (KGA and GAC). CB-839 had antiproliferative activity in a triple-negative breast cancer (TNBC) cell line, HCC-1806, that was associated with a marked decrease in glutamine consumption, glutamate production, oxygen consumption, and the steady-state levels of glutathione and several tricarboxylic acid cycle intermediates. In contrast, no antiproliferative activity was observed in an estrogen receptor-positive cell line, T47D, and only modest effects on glutamine consumption and downstream metabolites were observed. Across a panel of breast cancer cell lines, GAC protein expression and glutaminase activity were elevated in the majority of TNBC cell lines relative to receptor positive cells. Furthermore, the TNBC subtype displayed the greatest sensitivity to CB-839 treatment and this sensitivity was correlated with (i) dependence on extracellular glutamine for growth, (ii) intracellular glutamate and glutamine levels, and (iii) GAC (but not KGA) expression, a potential biomarker for sensitivity. CB-839 displayed significant antitumor activity in two xenograft models: as a single agent in a patient-derived TNBC model and in a basal like HER2 þ cell line model, JIMT-1, both as a single agent and in combination with paclitaxel. Together, these data provide a strong rationale for the clinical investigation of CB-839 as a targeted therapeutic in patients with TNBC and other glutamine-dependent tumors. Mol Cancer Ther; 13(4); 890-901. Ó2014 AACR.

The immunoproteasome, a distinct class of proteasome found predominantly in monocytes and lymphocytes, is known to shape the antigenic repertoire presented on class I major histocompatibility complexes (MHC-I). However, a specific role for the immunoproteasome in regulating other facets of immune responses has not been established. We describe here the characterization of PR-957, a selective inhibitor of low-molecular mass polypeptide-7 (LMP7, encoded by Psmb8), the chymotrypsin-like subunit of the immunoproteasome. PR-957 blocked presentation of LMP7-specific, MHC-I-restricted antigens in vitro and in vivo. Selective inhibition of LMP7 by PR-957 blocked production of interleukin-23 (IL-23) by activated monocytes and interferon-gamma and IL-2 by T cells. In mouse models of rheumatoid arthritis, PR-957 treatment reversed signs of disease and resulted in reductions in cellular infiltration, cytokine production and autoantibody levels. These studies reveal a unique role for LMP7 in controlling pathogenic immune responses and provide a therapeutic rationale for targeting LMP7 in autoimmune disorders.

Clinical studies with bortezomib have validated the proteasome as a therapeutic target for the treatment of multiple myeloma and non-Hodgkin's lymphoma. However, significant toxicities have restricted the intensity of bortezomib dosing.

Membrane-enveloped vesicles travel among the compartments of the cytoplasm of eukaryotic cells, delivering their specific cargo to programmed locations by membrane fusion. The pairing of vesicle v-SNAREs (soluble N-ethylmaleimide-sensitive factor attachment protein receptors) with target membrane t-SNAREs has a central role in intracellular membrane fusion. We have tested all of the potential v-SNAREs encoded in the yeast genome for their capacity to trigger fusion by partnering with t-SNAREs that mark the Golgi, the vacuole and the plasma membrane. Here we find that, to a marked degree, the pattern of membrane flow in the cell is encoded and recapitulated by its isolated SNARE proteins, as predicted by the SNARE hypothesis.

Carfilzomib is a proteasome inhibitor in clinical development that primarily targets the chymotrypsin-like (CT-L) subunits in both the constitutive proteasome (c20S) and the immunoproteasome (i20S). To investigate the impact of inhibiting the CT-L activity with carfilzomib, we set out to quantitate the levels of CT-L subunits ␤5 from the c20S and LMP7 from the i20S in normal and malignant hematopoietic cells. We found that the i20S is a major form of the proteasome expressed in cells of hematopoietic origin, including multiple myeloma (MM) CD138 ؉ tumor cells. Although specific inhibition of either LMP7 or ␤5 alone was insufficient to produce an antitumor response, inhibition of all proteasome subunits was cytotoxic to both hematologic tumor cells and peripheral blood mononuclear cells. However, selective inhibition of both ␤5 and LMP7 was sufficient to induce an antitumor effect in MM, non-Hodgkin lymphoma, and leukemia cells while minimizing the toxicity toward nontransformed cells. In MM tumor cells, CT-L inhibition alone was sufficient to induce proapoptotic sequelae, including proteasome substrate accumulation, Noxa and caspase 3/7 induction, and phospho-eIF2␣ suppression. These data support a hypothesis that hematologic tumor cells are uniquely sensitive to CT-L inhibition and provide a mechanistic understanding of the clinical safety profile and antitumor activity of proteasome inhibitors. (Blood.

Purpose: Bortezomib (Velcade), a dipeptide boronate 20S proteasome inhibitor and an approved treatment option for multiple myeloma, is associated with a treatment-emergent, painful peripheral neuropathy (PN) in more than 30% of patients. Carfilzomib, a tetrapeptide epoxyketone proteasome inhibitor, currently in clinical investigation in myeloma, is associated with low rates of PN. We sought to determine whether PN represents a target-mediated adverse drug reaction (ADR).Experimental Design: Neurodegenerative effects of proteasome inhibitors were assessed in an in vitro model utilizing a differentiated neuronal cell line. Secondary targets of both inhibitors were identified by a multifaceted approach involving candidate screening, profiling with an activity-based probe, and database mining. Secondary target activity was measured in rats and patients receiving both inhibitors.Results: Despite equivalent levels of proteasome inhibition, only bortezomib reduced neurite length, suggesting a nonproteasomal mechanism. In cell lysates, bortezomib, but not carfilzomib, significantly inhibited the serine proteases cathepsin G (CatG), cathepsin A, chymase, dipeptidyl peptidase II, and HtrA2/Omi at potencies near or equivalent to that for the proteasome. Inhibition of CatG was detected in splenocytes of rats receiving bortezomib and in peripheral blood mononuclear cells derived from bortezomib-treated patients. Levels of HtrA2/Omi, which is known to be involved in neuronal survival, were upregulated in neuronal cells exposed to both proteasome inhibitors but was inhibited only by bortezomib exposure.Conclusion: These data show that bortezomib-induced neurodegeneration in vitro occurs via a proteasome-independent mechanism and that bortezomib inhibits several nonproteasomal targets in vitro and in vivo, which may play a role in its clinical ADR profile. Clin Cancer Res; 17(9); 2734-43. Ó2011 AACR.

. We find that the isolated core of a SNARE complex efficiently fuses artificial bilayers and does so faster than full length SNAREs. Unexpectedly, a dramatic increase in speed results from removal of the N-terminal domain of the t-SNARE syntaxin, which does not affect the rate of assembly of v-t SNARES. In the absence of this negative regulatory domain, the half-time for fusion of an entire population of lipid vesicles by isolated SNARE cores (Ϸ10 min) is compatible with the kinetics of fusion in many cell types. Many genetic and biochemical experiments have implicated SNAREs in the overall process of membrane fusion (1-6). The finding that isolated SNARE proteins can efficiently fuse lipid bilayers directly establishes that they are the basic machinery that merges membranes (7), a conclusion recently confirmed in an elegant study using permeabilized cells (8) and by the demonstration of contents mixing during SNARE-dependent fusion [see the accompanying paper by Nickel et al. (9)]. This principle is underscored by the internal architecture of the SNARE complex, whose ''core'' consists of four parallel ␣-helices packed into a single bundle with their membrane anchors emerging together from one end of the assembled SNARE complex (10-14).The structure of the cytoplasmic core domain of a SNARE complex is reminiscent of the structure of the extracellularly localized core region of many viral fusion proteins in what is thought to correspond to a postfusogenic conformation (15,16). This suggests a general principle for membrane fusion, in which pin-like structures bridging two membranes promote their fusion. However, no core complex, whether cellular or viral, has actually been shown to be fusogenic. Here, we report such evidence with a core domain of SNARE proteins. Materials and MethodsPlasmid Construction, Protein Expression, and Purification. The following v-SNARE and t-SNARE complexes were bacterially expressed and purified by nickel affinity chromatography: VA MP2His 6 ; syntaxin1͞His 6 SNA P-25 (SYN͞SNA P-25); thrombin-cleavable syntaxin1͞His 6 SNAP-25 (tcSYN͞SNAP-25); syntax in1͞thrombin-cleavableHis 6 SNA P-25 (SYN͞ tcSNAP-25); and thrombin-cleavable syntaxin1͞thrombin-cleavableHis 6 SNAP-25 (tcSYN͞tcSNAP-25). For detailed information, see the supplemental material on the PNAS web site, www.pnas.org.Protein Reconstitution into Liposomes and Thrombin Cleavage of t-SNARE Liposomes. Both VAMP2 and t-SNARE complexes were reconstituted into liposomes as described (7). Once proteoliposomes were harvested, tcSYN͞SNAP-25 liposomes were first treated with human thrombin (Sigma, catalogue number T-1063) at 0.02 units͞l for 2 hours at room temperature and subsequently were inhibited with 2 mM 4-(2-aminoethyl)benzenesulfonyl f luoride (Calbiochem). SYN͞tcSNA P-25, tcSYN͞ tcSNAP-25, or SYN͞SNAP-25 containing proteoliposomes were treated with 0.04 units͞l thrombin (Sigma) for 4 hours at 37°C and subsequently were inhibited with 2 mM 4-(2-aminoethyl)benzenesulfonyl fluoride. As a control, thrombin was preinactivated by fir...