In mitosis, the anaphase-promoting complex (APC) regulates the onset of sister-chromatid separation and exit from mitosis by mediating the ubiquitination and degradation of the securin protein and mitotic cyclins. With the use of a baculoviral expression system, we have reconstituted the ubiquitin ligase activity of human APC. In combination with Ubc4 or UbcH10, a heterodimeric complex of APC2 and APC11 is sufficient to catalyze the ubiquitination of human securin and cyclin B1. However, the minimal APC2/11 ubiquitin ligase module does not possess substrate specificity, because it also ubiquitinates the destruction box deletion mutants of securin and cyclin B1. Both APC11 and UbcH10 bind to the C-terminal cullin homology domain of APC2, whereas Ubc4 interacts with APC11 directly. Zn 2ϩ -binding and mutagenesis experiments indicate that APC11 binds Zn 2ϩ at a 1:3 M ratio. Unlike the two Zn 2ϩ ions of the canonical RING-finger motif, the third Zn 2ϩ ion of APC11 is not essential for its ligase activity. Surprisingly, with Ubc4 as the E2 enzyme, Zn 2ϩ ions alone are sufficient to catalyze the ubiquitination of cyclin B1. Therefore, the Zn 2ϩ ions of the RING finger family of ubiquitin ligases may be directly involved in catalysis.

The 26 S proteasome is a large protease complex that catalyzes the degradation of both native and misfolded proteins. These proteins are known to interact with PA700, the regulatory subcomplex of the 26 S proteasome, via a covalently attached polyubiquitin chain. Here we provide evidence for an additional ubiquitinindependent mode of substrate recognition by PA700. PA700 prevents the aggregation of three incompletely folded, nonubiquitinated substrates: the ⌬F-508 mutant form of cystic fibrosis transmembrane regulator, nucleotide binding domain 1, insulin B chain, and citrate synthase. This function does not require ATP hydrolysis. The stoichiometry required for this function, the effect of PA700 on the lag phase of aggregation, and the temporal specificity of PA700 in this process all indicate that PA700 interacts with a subpopulation of non-native conformations that is either particularly aggregationprone or nucleates misassociation reactions. The inhibition of off-pathway self-association reactions is also reflected in the ability of PA700 to promote refolding of citrate synthase. These results provide evidence that, in addition to binding polyubiquitin chains, PA700 contains a site(s) that recognizes and interacts with misfolded or partially denatured polypeptides. This feature supplies an additional level of substrate specificity to the 26 S proteasome and a means by which substrates are maintained in a soluble state until refolding or degradation is complete.

Cellular senescence is widely believed to play a key role in tumor suppression, but the molecular pathways that regulate senescence are only incompletely understood. By using a secretome proteomics approach, we identified insulin-like growth factor binding protein 3 (IGFBP3) as a secreted mediator of breast cancer senescence upon chemotherapeutic drug treatment. The senescenceinducing activity of IGFBP3 is inhibited by tissue-type plasminogen activator-mediated proteolysis, which is counteracted by plasminogen activator inhibitor 1 (PAI-1), another secreted mediator of senescence. We demonstrate that IGFBP3 is a critical downstream target of PAI-1-induced senescence. These results suggest a role for an extracellular cascade of secreted proteins in the regulation of cellular senescence.chemotherapy | isotope-coded affinity tag | protease | protease inhibitor | protein secretion C ellular senescence was originally described as irreversible proliferation arrest that limits the proliferation of human primary cells in culture (1). This phenomenon, "replicative senescence," is caused by progressive shortening of telomeres. In addition, a variety of stressful or oncogenic stimuli can elicit a senescence response. These include DNA damage, oxidative stress, and expression of certain oncogenes such as ras and raf (2, 3). Accumulating evidence suggests that cellular senescence plays important roles in tumor suppression and organismal aging, but how senescence is regulated is largely unknown.In addition to irreversible arrest of proliferation, senescent cells often display common phenotypes such as enlarged, flattened morphology, increased lysosome biogenesis, decreased protein synthesis and degradation, senescence-associated β-gal (SA β-gal) activity, and increased expression of cell cycle inhibitors (2, 3). Further, recent studies have begun to uncover profound alterations in protein secretion from senescent cells, which is collectively called the senescence-associated secretory phenotype (SASP) (4, 5) or senescence-messaging secretome (SMS) (6). These include increased secretion of inflammatory cytokines such as interleukins and chemokines, proteases, and growth regulators. These SASP or SMS factors may recruit immune cells for clearance of senescent cells, affect the architecture or function of surrounding tissues, modulate tumor progression, and contribute to aging and age-related diseases.Most cancer cells express telomerase and do not undergo replicative senescence. However, cancer cells from a variety of tissues undergo senescence upon treatment with chemotherapeutic drugs or ionizing radiation (7,8). Senescent cancer cells are characterized by proliferation arrest, flat and enlarged morphology, and SA β-gal activity, which is similar to the senescence phenotype of primary cells. A previous report demonstrated that naive MCF-7 breast cancer cells undergo senescence upon exposure to conditioned medium from senescent MCF-7 cells induced to senesce by treatment with doxorubicin, a widely used chemotherapeutic drug for...

We have identified, purified, and characterized three subcomplexes of PA700, the 19 S regulatory complex of the 26 S proteasome. These subcomplexes (denoted PS-1, PS-2, and PS-3) collectively account for all subunits present in purified PA700 but contain no overlapping components or significant levels of non-PA700 proteins. Each subcomplex contained two of the six AAA subunits (Rpt1-6) that form the binding interface of PA700 with the 20 S proteasome, the protease component of the 26 S proteasome. Unlike intact PA700, no individual PA700 subcomplex displayed ATPase activity or proteasome activating activity. However, both activities were manifested by ATP-dependent in vitro reconstitution of PA700 from the subcomplexes. We exploited functional reconstitution to define and distinguish roles of different PA700 subunits in PA700 function by selective alteration of subunits within individual subcomplexes prior to reconstitution. Carboxypeptidase treatment of either PS-2 or PS-3, subcomplexes containing specific Rpt subunits previously shown to have important roles in 26 S proteasome assembly and activation, inhibited these processes but did not affect PA700 reconstitution or ATPase activity. Thus, the intact C termini of both subunits are required for 26 S proteasome assembly and activation but not for PA700 reconstitution. Surprisingly, carboxypeptidase treatment of PS-1 also inhibited 26 S proteasome assembly and activation upon reconstitution with untreated PS-2 and PS-3. These results suggest a previously unidentified role for other PA700 subunits in 26 S proteasome assembly and activation. Our results reveal relative structural and functional relationships among the AAA subunits of PA700 and new insights about mechanisms of 26 S proteasome assembly and activation.The 26 S proteasome is a 2,500,000-Da protease complex that degrades polyubiquitylated proteins by an ATP-dependent mechanism (1, 2). The biochemical processes required for this function are divided between two subcomplexes that compose the holoenzyme (3, 4). The first, called 20 S proteasome or core particle, is a 700,000-Da complex that catalyzes peptide bond hydrolysis (5). The second, called PA700 or 19 S regulatory particle, is a 700,000-Da complex that mediates multiple aspects of proteasome function related to initial binding and subsequent delivery of substrates to the catalytic sites of the 20 S proteasome (6). The 20 S proteasome is composed of 28 subunits representing the products of 14 genes arranged in four axially stacked heteroheptameric rings (7,8). Each of the two center ␤ rings contains three different protease subunits that utilize N-terminal threonine residues as catalytic nucleophiles (5,8,9). These residues line an interior lumen formed by the stacked rings and thus are sequestered from interaction with substrates by a shell of 20 S proteasome subunits. PA700 is composed of 20 different subunits. Six of these subunits, termed Rpt1-6, are AAA 2 (ATPases Associated with various cellular Activities) family members that confer ATPase ...

The extracellular matrix (ECM) is a critical tissue component, providing structural support as well as important regulatory signaling cues to govern cellular growth, metabolism, and differentiation. The study of ECM proteins, however, is hampered by the low solubility of ECM components in common solubilizing reagents. ECM proteins are often not detected during proteomics analyses using unbiased approaches due to solubility issues and relatively low abundance compared to highly abundant cytoplasmic and mitochondrial proteins. Decellularization has become a common technique for ECM protein-enrichment and is frequently used in engineering studies. Solubilizing the ECM after decellularization for further proteomic examination has not been previously explored in depth. In this study, we describe testing of a series of protocols that enabled us to develop a novel optimized strategy for the enrichment and solubilization of ECM components. Following tissue decellularization, we use acid extraction and enzymatic deglycosylation to facilitate resolubilization. The end result is the generation of three fractions for each sample: soluble components, cellular components, and an insoluble ECM fraction. These fractions, developed in mass spectrometry-compatible buffers, are amenable to proteomics analysis. The developed protocol allows identification (by mass spectrometry) and quantification (by mass spectrometry or immunoblotting) of ECM components in tissue samples. Biological significance The study of extracellular matrix (ECM) proteins in pathological and non-pathological conditions is often hampered by the low solubility of ECM components in common solubilizing reagents. Additionally, ECM proteins are often not detected during global proteomic analyses due to their relatively low abundance compared to highly abundant cytoplasmic and mitochondrial proteins. In this manuscript we describe testing of a series of protocols that enabled us to develop a final novel optimized strategy for the enrichment and solubilization of ECM components. The end result is the generation of three fractions for each sample: soluble components, cellular components, and an insoluble ECM fraction. By analysis of each independent fraction, differences in protein levels can be detected that in normal conditions would be masked. These fractions are amenable to mass spectrometry analysis to identify and quantify ECM components in tissue samples. The manuscript places a strong emphasis on the immediate practical relevance of the method, particularly when using mass spectrometry approaches; additionally, the optimized method was validated and compared to other methodologies described in the literature.