Binding of activated α 2 -macroglobulin to GRP78 on the surface of human prostate cancer cells promotes proliferation by activating signaling cascades. Autoantibodies directed against the activated α 2 -macroglobulin binding site in the NH 2 -terminal domain of GRP78 are receptor agonists, and their presence in the sera of cancer patients is a poor prognostic indicator. We now show that antibodies directed against the GRP78 COOH-terminal domain inhibit [3 H]thymidine uptake and cellular proliferation while promoting apoptosis as measured by DNA fragmentation, Annexin V assay, and clonogenic assay. These antibodies are receptor antagonists blocking autophosphorylation and activation of GRP78. Using 1-LN and DU145 prostate cancer cell lines and A375 melanoma cells, which express GRP78 on their cell surface, we show that antibodies directed against the COOH-terminal domain of GRP78 up-regulate the tumor suppressor protein p53. By contrast, antibody directed against the NH 2

The halophilic archaeon Haloferax volcanii produces three different proteins (␣1, ␣2, and ␤) that assemble into at least two 20S proteasome isoforms. This work reports the cloning and sequencing of two H. volcanii proteasome-activating nucleotidase (PAN) genes (panA and panB). The deduced PAN proteins were 60% identical with Walker A and B motifs and a second region of homology typical of AAA ATPases. The most significant region of divergence was the N terminus predicted to adopt a coiled-coil conformation involved in substrate recognition. Of the five proteasomal proteins, the ␣1, ␤, and PanA proteins were the most abundant. Differential regulation of all five genes was observed, with a four-to eightfold increase in mRNA levels as cells entered stationary phase. In parallel with this mRNA increase, the protein levels of PanB and ␣2 increased severalfold during the transition from exponential growth to stationary phase, suggesting that these protein levels are regulated at least in part by mechanisms that control transcript levels. In contrast, the ␤ and PanA protein levels remained relatively constant, while the ␣1 protein levels exhibited only a modest increase. This lack of correlation between the mRNA and protein levels for ␣1, ␤, and PanA suggests posttranscriptional mechanisms are involved in regulating the levels of these major proteasomal proteins. Together these results support a model in which the cell regulates the ratio of the different 20S proteasome and PAN proteins to modulate the structure and ultimately the function of this central energy-dependent proteolytic system.Proteasomes are large nanocompartments within the cell that catalyze the energy-dependent hydrolysis of proteins. The catalytic core particle (CP) responsible for peptide bond hydrolysis is a 20S cylinder of four stacked heptameric rings which is ubiquitous in archaea, eucaryotes, and actinomycetes (19,22). The outer and inner rings of the CP are formed by proteins of related ␣ and ␤ superfamilies, respectively (6). A variety of components associate with the CP; most notable are the 19S cap regulatory particles (RP), which together with the CP form eucaryal 26S proteasomes. The RP of yeast can be separated into two multisubunit substructures: the lid and base domains. The base includes six Rpt (regulatory particle triple-A type I ATPase) subunits and not only exhibits chaperone activity (4) but also facilitates the energy-dependent degradation of globular proteins by 20S proteasomes (10). These Rpt subunits are closely related to archaeal proteasome-activating nucleotidase (PAN) proteins, of which only the Methanocaldococcus jannaschii Pan purified from recombinant Escherichia coli has been characterized (2, 38, 44). Substrate binding to this AAA ATPase activates the hydrolysis of nucleotides (e.g., ATP), which subsequently promotes the unfolding of substrates, opening of the 20S proteasome axial gate(s), and apparent translocation of substrate to the internal proteolytic chamber of 20S proteasomes.Multicellular organisms synthesize...

Haloferax volcanii, a halophilic archaeon, synthesizes three different proteins (␣1, ␣2, and ␤) which are classified in the 20S proteasome superfamily. The ␣1 and ␤ proteins alone form active 20S proteasomes; the role of ␣2, however, is not clear. To address this, ␣2 was synthesized with an epitope tag and purified by affinity chromatography from recombinant H. volcanii. The ␣2 protein copurified with ␣1 and ␤ in a complex with an overall structure and peptide-hydrolyzing activity comparable to those of the previously described ␣1-␤ proteasome. Supplementing buffers with 10 mM CaCl 2 stabilized the halophilic proteasomes in the absence of salt and enabled them to be separated by native gel electrophoresis. This facilitated the discovery that wild-type H. volcanii synthesizes more than one type of 20S proteasome. Two 20S proteasomes, the ␣1-␤ and ␣1-␣2-␤ proteasomes, were identified during stationary phase. Cross-linking of these enzymes, coupled with available structural information, suggested that the ␣1-␤ proteasome was a symmetrical cylinder with ␣1 rings on each end. In contrast, the ␣1-␣2-␤ proteasome appeared to be asymmetrical with homo-oligomeric ␣1 and ␣2 rings positioned on separate ends. Inter-␣-subunit contacts were only detected when the ratio of ␣1 to ␣2 was perturbed in the cell using recombinant technology. These results support a model that the ratio of ␣ proteins may modulate the composition and subunit topology of 20S proteasomes in the cell.Proteasomes are large, energy-dependent proteases. These enzyme structures form nanocompartments within the cell (2, 27) that degrade proteins into oligopeptides of 3 to 30 amino acids in length by processive hydrolysis (1, 24). The catalytic core responsible for this proteolytic activity is a 20S particle, universally distributed among the Archaea, Eucarya, and grampositive actinomycetes (10, 12). The 20S proteasome particle (11 to 12 nm in diameter and 15 nm in length) is a cylindrical bundle of four-stacked, heptameric rings. This barrel-shaped structure includes a central channel with narrow (1.3 nm) openings on each end which limits substrate access (32). Each opening is connected to a central chamber responsible for the hydrolysis of peptide bonds (20,21,25). "Unfoldases" such as the eucaryal 19S cap and archaeal PAN protein associate with 20S proteasomes and stimulate the energy-dependent degradation of proteins (17,34,37,39).The subunits that form 20S proteasomes have been classified into two related superfamilies (␣ and ␤) (9). The ␣ proteins form the outer rings (18) and are required for the ␤ proteins to be processed during formation of inner rings which harbor the active-site N-terminal threonine (13,25,26,31,38). The number of subunits forming 20S proteasomes varies. Many lower eucaryotes such as yeast produce a single symmetrical 20S proteasome of 14 different subunits (i.e., two copies each of ␣1 to ␣7 and ␤1 to ␤7) (20). Higher eucaryotes express additional subunits (e.g., ␤1i, ␤2i, ␤5i) that form auxiliary 20S proteasomes (e.g., the immuno...

Both the voltage-dependent anion channel and the glucoseregulated protein 78 have been identified as plasminogen kringle 5 receptors on endothelial cells. In this study, we demonstrate that kringle 5 binds to a region localized in the N-terminal domain of the glucose-regulated protein 78, whereas microplasminogen does so through the C-terminal domain of the glucose-regulated protein 78. Both plasminogen fragments induce Ca 2؉ signaling cascades; however, kringle 5 acts through voltage-dependent anion channel and microplasminogen does so via the glucose-regulated protein 78. Because trafficking of voltage-dependent anion channel to the cell surface is associated with heat shock proteins, we investigated a possible association between voltage-dependent anion channel and glucose-regulated protein 78 on the surface of 1-LN human prostate tumor cells. We demonstrate that these proteins co-localize, and changes in the expression of the glucoseregulated protein 78 affect the expression of voltage-dependent anion channel. To differentiate the functions of these receptor proteins, either when acting singly or as a complex, we employed human hexokinase I as a specific ligand for voltage-dependent anion channel, in addition to kringle 5. We show that kringle 5 inhibits 1-LN cell proliferation and promotes caspase-7 activity by a mechanism that requires binding to cell surface voltage-dependent anion channel and is inhibited by human hexokinase I. Plasminogen (Pg)2 is a single-chain 92-kDa glycoprotein containing 791 amino acid residues (1, 2). There are seven distinct domains in the Pg molecule: an N-terminal peptide (NTP), five kringle domains, and a serine proteinase domain (3). The kringle domains, each comprising ϳ80 amino acids, are double-looped disulfide-bonded structures that contain the lysinebinding sites (LBS) responsible for Pg binding to extracellular matrix molecules (4) and cell receptors (5, 6). In addition to the LBS, Pg also has three benzamidine binding sites (BBS), one in K5, a second in the active site of the proteinase domain, and a third unknown site in the Pg proteinase domain (7).Pg is the precursor of angiostatins, a group of anti-angiogenic molecules, consisting of kringles 1-3 (K1-3), 1-4, and 1-5, as well as the single kringles 1 (K1), 2, 3, and 5, but not K4 (8 -10). We have extensively studied the functions of K1-3 and K5 on both endothelial and prostate tumor cell lines (11,12). We reported that K5 binding to the voltage-dependent anion channel (VDAC) on the surface of human umbilical vein endothelial cells (HUVEC) interfered with mechanisms controlling the regulation of intracellular Ca 2ϩ producing a decrease in intracellular pH (11). A recent study also suggests, that K5 binds with high affinity to surface-expressed glucose-regulated protein 78 (GRP78) (13). Both VDAC and GRP78 are expressed on the cell surface in lipid rafts (14, 15). Because trafficking of VDAC to the cell membrane is associated with the heat shock proteins GRP75 and HSP70 (16, 17), we hypothesized a possible associatio...