Degradation of Endocytosed Gap Junctions by Autophagosomal and Endo-/lysosomal Pathways: A Perspective

Gap junctions (GJs) are composed of tens to many thousands of double-membrane spanning GJ channels that cluster together to form densely packed channel arrays (termed GJ plaques) in apposing plasma membranes of neighboring cells. In addition to providing direct intercellular communication (GJIC, their hallmark function), GJs, based on their characteristic double-membrane-spanning configuration, likely also significantly contribute to physical cell-to-cell adhesion. Clearly, modulation (up-/down-regulation) of GJIC and of physical cell-to-cell adhesion is as vitally important as the basic ability of GJ formation itself. Others and we have previously described that GJs can be removed from the plasma membrane via the internalization of entire GJ plaques (or portions thereof) in a cellular process that resembles clathrin-mediated endocytosis. GJ endocytosis results in the formation of double-membrane vesicles [termed annular gap junctions (AGJs) or connexosomes] in the cytoplasm of one of the coupled cells. Four recent independent studies, consistent with earlier ultrastructural analyses, demonstrate the degradation of endocytosed AGJ vesicles via autophagy. However, in TPA-treated cells others report degradation of AGJs via the endo-/lysosomal degradation pathway. Here we summarize evidence that supports the concept that autophagy serves as the cellular default pathway for the degradation of internalized GJs. Furthermore, we highlight and discuss structural criteria that seem required for an alternate degradation via the endo-/lysosomal pathway.     

Autophagic Degradation of NBR1 Restricts Metastatic Outgrowth during Mammary Tumor Progression

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Addition of Aromatic Substrates Restores Trichloroethylene Degradation Activity in Pseudomonas putida F1

The rate of trichloroethylene (TCE) degradation by toluene dioxygenase (TDO) in resting cells of Pseudomonas putida F1 gradually decreased and eventually stopped within 1.5 h, as in previous reports. However, the subsequent addition of toluene, which is the principal substrate of TDO, resulted in its immediate degradation without a lag phase. After the consumption of toluene, degradation of TCE restarted at a rate similar to its initial degradation, suggesting that this degradation was mediated by TDO molecules that were present before the cessation of TCE degradation. The addition of benzene and cumene, which are also substrates of TDO, also caused restoration of TCE degradation activity: TCE was degraded simultaneously with cumene, and a larger amount of TCE was degraded after cumene was added than after toluene or benzene was added. But substrates that were expected to supply the cells with NADH or energy did not restore TCE degradation activity. This cycle of pseudoinactivation and restoration of TCE degradation was observed repeatedly without a significant decrease in the number of viable cells, even after six additions of toluene spread over 30 h. The results obtained in this study demonstrate a new type of restoration of TCE degradation that has not been previously reported.     

Engineering Synthetic Adaptors and Substrates for Controlled ClpXP Degradation

Facile control of targeted intracellular protein degradation has many potential uses in basic science and biotechnology. One promising approach to this goal is to redesign adaptor proteins, which can regulate proteolytic specificity by tethering substrates to energy-dependent AAA+ proteases. Using the ClpXP protease, we have probed the minimal biochemical functions required for adaptor function by designing and characterizing variant substrates, adaptors, and ClpX enzymes. We find that substrate tethering mediated by heterologous interaction domains and a small bridging molecule mimics substrate delivery by the wild-type system. These results show that simple tethering is sufficient for synthetic adaptor function. In our engineered system, tethering and proteolysis depend on the presence of the macrolide rapamycin, providing a foundation for engineering highly specific degradation of target proteins in cells. Importantly, this degradation is regulated by a small molecule without the need for new adaptor or enzyme biosynthesis.Targeted proteolytic degradation plays important roles in protein quality control and in regulating cellular circuitry in organisms ranging from bacteria to humans (1–4). In some instances, substrates are recognized directly by a protease enzyme via a degradation tag (Fig. 1A) (see Refs. 5 and 6). In other cases, adaptor proteins or multiple types of substrate sequences are also required to ensure efficient degradation (Fig. 1, B and C) (see Refs. 7 and 8).Open in a separate windowFIGURE 1.A, the ClpX component of the ClpXP protease recognizes some substrates via a degradation tag, denatures the substrate, and then translocates the unfolded protein into ClpP for degradation. B, adaptor-assisted binding of a substrate to ClpXP. C, self-tethering of a substrate to ClpXP.Experimentally induced degradation can be used as a tool to probe the role of specific proteins in cellular processes. For example, a protein that is normally stable can be modified to make its degradation conditionally dependent on the presence of an adaptor, allowing studies of the consequences of depletion after induction of adaptor synthesis (7, 9). Such systems complement methods, such as RNA interference, that rely upon repressing biosynthesis of the target protein but offer significant advantages when rapid depletion of otherwise long-lived proteins is the goal (10–12). We are interested in engineering synthetic adaptor systems to control targeted intracellular degradation.ClpXP is a AAA+ protease present in bacteria and mitochondria that consists of two components, ClpX and ClpP. Hexamers of ClpX recognize degradation tags in specific substrate proteins, unfold them in a reaction that requires ATP hydrolysis, and then use additional cycles of ATP hydrolysis to translocate the unfolded polypeptide into an interior chamber of ClpP, where proteolysis takes place (see Fig. 1A). The simplest way in which an adaptor could stimulate degradation is by tethering a specific substrate to a protease, thereby increasing its effective concentration and facilitating proteolysis (see Fig. 1B; for review, see Ref. 3). The SspB adaptor, for example, appears to function by this mechanism. SspB enhances ClpXP degradation of certain substrates, including N-RseA and proteins bearing the ssrA-degradation tag (2, 8, 13). ClpXP degrades these substrates in the absence of SspB, but K_m for degradation is substantially lower when this adaptor is present. Two features of SspB are consistent with a tethering mechanism. It has a substrate-binding domain with a groove that binds a portion of the ssrA tag or a sequence in N-RseA, and it contains a flexible C-terminal extension terminating with a peptide motif (XB) that binds to the N-terminal domain of ClpX (14–19). Mutations that prevent SspB binding to ClpX or block substrate binding to SspB eliminate stimulation of degradation (13, 16, 20).It has not been rigorously established, however, that tethering per se is sufficient for the activity of any adaptor. Based on biochemical experiments, for instance, Thibault et al. (21) proposed that the adaptor activity of SspB is mediated, in part, by its ability to direct the movement of the N-terminal domains of ClpX, and thereby to regulate the delivery of tagged substrates to ClpXP. For some adaptors, tethering of the substrate to the protease is not sufficient for degradation. For example, the ClpS adaptor tethers N-end rule substrates to the AAA+ ClpAP protease (22–24), but some ClpS mutants mediate efficient substrate tethering to ClpAP without facilitating degradation (25). In such cases, more complicated transactions between the adaptor and the protease appear to be needed to ensure that the substrate is properly delivered to the protease. Moreover, in some instances, adaptors play roles in substrate delivery but are also required for assembly of the active protease (26).The studies reported here were motivated by two major goals. First, we wished to test if a completely synthetic adaptor system could be used to regulate substrate degradation. Second, we sought to design a proteolysis system that could be controlled by the presence or absence of a small molecule. To define the minimal biochemical properties required for adaptor-protein function, we engineered and characterized synthetic variants of adaptors, substrates, and the ClpXP protease. We reasoned that if specialized interactions between SspB and the N-terminal domain of ClpX were a requisite part of substrate delivery, then replacing either component would preclude efficient degradation. By contrast, we found that rapid degradation of an otherwise poor substrate was possible in the absence of SspB and the N-domain as long as substrate·enzyme tethering was maintained by other interaction domains. These results show that tethering alone is sufficient for synthetic- adaptor function. We were also able to control degradation in vitro and in vivo using systems in which a small molecule, rapamycin, drives assembly of tethered proteolytic complexes. Thus, targeted degradation can be engineered to depend, in a conditional fashion, on the presence of a small molecule. In principle, degradation under small molecule control has many of the advantages of chemical genetics (27), but should be even simpler and more widely applicable as a method of functional inhibition. In addition, controlling degradation in this fashion is possible even when biosynthesis of new macromolecules is precluded.     

Subcellular Fractionation Analysis of the Extraction of Ubiquitinated Polytopic Membrane Substrate during ER-Associated Degradation

During ER-associated degradation (ERAD), misfolded polytopic membrane proteins are ubiquitinated and retrotranslocated to the cytosol for proteasomal degradation. However, our understanding as to how polytopic membrane proteins are extracted from the ER to the cytosol remains largely unclear. To better define the localization and physical properties of ubiquitinated polytopic membrane substrates in vivo, we performed subcellular fractionation analysis of Ste6*, a twelve transmembrane protein that is ubiquitinated primarily by Doa10 E3 ligase in yeast. Consistent with previous in vitro studies, ubiquitinated Ste6* was extracted from P20 (20,000 g pellet) fraction to S20 (20,000 g supernatant) fraction in a Cdc48/p97-dependent manner. Similarly, Ubx2p, which recruits Cdc48/p97 to the ER, facilitated the extraction of Ste6*. By contrast, lipid droplet formation, which was suggested to be dispensable for the degradation of Hrd1-substrates in yeast, was not required for the degradation of Ste6*. Intriguingly, we found that ubiquitinated Ste6* in the S20 fraction could be enriched by further centrifugation at 100,000 g. Although it is currently uncertain whether ubiquitinated Ste6* in P100 fraction is completely free from any lipids, membrane flotation analysis suggested the existence of two distinct populations of ubiquitinated Ste6* with different states of membrane association. Together, these results imply that ubiquitinated Ste6* may be sequestered into a putative quality control sub-structure by Cdc48/p97. Fractionation assays developed in the present study provide a means to further dissect the ill-defined post-ubiquitination step during ERAD of polytopic membrane substrates.     

The G3BP1-Family-USP10 Deubiquitinase Complex Rescues Ubiquitinated 40S Subunits of Ribosomes Stalled in Translation from Lysosomal Degradation

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Ubiquitinated annexin A2 is enriched in the cytoskeleton fraction

Annexin A2 is a multifunctional protein and its cellular functions are regulated by post-translational modifications and ligand binding. When purified from porcine intestinal mucosa and transformed mouse Krebs II cells, SDS-PAGE revealed high-molecular-mass forms in addition to the 36 kDa protomer. These forms were identified as poly-/multi-ubiquitin conjugates of annexin A2, and ubiquitination represents a novel post-translational modification of this protein. Subcellular fractionation of mouse Krebs II cells revealed an enrichment of annexin A2-ubiquitin conjugates in the Triton X-100 resistant cytoskeleton fraction, suggesting that ubiquitinated annexin A2 may have a role associated with its function as an actin-binding protein.     

Deterministic Three-Half-Order Kinetic Model for Microbial Degradation of Added Carbon Substrates in Soil

The kinetics of mineralization of carbonaceous substrates has been explained by a deterministic model which is applicable to either growth or nongrowth conditions in soil. The mixed-order nature of the model does not require a priori decisions about reaction order, discontinuity period of lag or stationary phase, or correction for endogenous mineralization rates. The integrated equation is simpler than the integrated form of the Monod equation because of the following: (i) only two, rather than four, interdependent constants have to be determined by nonlinear regression analysis, (ii) substrate or product formation can be expressed explicitly as a function of time, (iii) biomass concentration does not have to be known, and (iv) the required initial estimate for the nonlinear regression analysis can be easily obtained from a linearized form rather than from an interval estimate of a differential equation. ¹⁴CO₂ evolution data from soil have been fitted to the model equation. All data except those from irradiated soil gave better fits by residual sum of squares (RSS) by assuming growth in soil was linear (RSS = 0.71) as opposed to exponential (RSS = 2.87). The underlying reasons for growth (exponential versus linear), no growth, and relative degradation rates of substrates are consistent with the basic mechanisms from which the model is derived.     

Proteome-wide Analysis Reveals Substrates of E3 Ligase RNF146 Targeted for Degradation

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Highlights

• •Proteome analyses reveal RNF146 and TNKS1/2 substrates targeted for degradation.
• •RNF146 KO and TNKS1/2 DKO cells display significantly different proteomes.
• •RNF146 has both TNKS-dependent and -independent substrates.

     

Epsin 1 is Involved in Recruitment of Ubiquitinated EGF Receptors into Clathrin-Coated Pits

Epsin consists of an epsin NH₂-terminal homology domain that promotes interaction with phospholipids, several AP-2-binding sites, two clathrin-binding sequences and several Eps15 homology domain-binding motifs. Epsin additionally possesses ubiquitin-interacting motifs (UIMs) and has been demonstrated to bind ubiquitinated cargo. We therefore investigated whether epsin promoted clathrin-mediated endocytosis of the ubiquitinated EGF receptor (EGFR). By immunoprecipitation, we found that epsin 1 interacted with ubiquitinated EGFR and that functional UIMs were essential for complex formation. Furthermore, RNA interference-mediated knockdown of epsin 1 was found to inhibit internalization of the EGFR, while having no effect on endocytosis of the transferrin receptor. Additionally, upon knockdown of epsin 1, translocation of the EGFR to central parts of clathrin-coated pits was inhibited. This supports the contention that epsin 1 promotes endocytosis of the ubiquitinated EGFR.     

Crystal structure of the PB1 domain of NBR1

The scaffold protein NBR1 is involved in signal transmission downstream of the serine/protein kinase from the giant muscle protein titin. Its N-terminal Phox and Bem1p (PB1) domain plays a critical role in mediating protein-protein interactions with both titin kinase and with another scaffold protein, p62. We have determined the crystal structure of the PB1 domain of NBR1 at 1.55A resolution. It reveals a type-A PB1 domain with two negatively charged residue clusters. We provide a structural perspective on the involvement of NBR1 in the titin kinase signalling pathway.     

Role of Quinolinate Phosphoribosyl Transferase in Degradation of Phthalate by Burkholderia cepacia DBO1

Two distinct regions of DNA encode the enzymes needed for phthalate degradation by Burkholderia cepacia DBO1. A gene coding for an enzyme (quinolinate phosphoribosyl transferase) involved in the biosynthesis of NAD+ was identified between these two regions by sequence analysis and functional assays. Southern hybridization experiments indicate that DBO1 and other phthalate-degrading B. cepacia strains have two dissimilar genes for this enzyme, while non-phthalate-degrading B. cepacia strains have only a single gene. The sequenced gene was labeled ophE, due to the fact that it is specifically induced by phthalate as shown by lacZ gene fusions. Insertional knockout mutants lacking ophE grow noticeably slower on phthalate while exhibiting normal rates of growth on other substrates. The fact that elevated levels of quinolinate phosphoribosyl transferase enhance growth on phthalate stems from the structural similarities between phthalate and quinolinate: phthalate is a competitive inhibitor of this enzyme and the phthalate catabolic pathway cometabolizes quinolinate. The recruitment of this gene for growth on phthalate thus gives B. cepacia an advantage over other phthalate-degrading bacteria in the environment.     

Reverse Genetic Identification of CRN1 and its Distinctive Role in Chlorophyll Degradation in Arabidopsis

Recent identification of NYE1/SGR1 brought up a new era for the exploration of the regulatory mechanism of Chlorophyll (Chl) degradation.Cluster analysis of senescence associated genes with putative chloroplast targeting sequences revealed several genes sharing a similar expression pattern with NYE1.Further characterization of available T-DNA insertion lines led to the discovery of a novel stay-green gene CRN1 ((C)o-(r)egulated with (N)YE1).Chl breakdown was significantly restrained in crn1-1 under diversified senescence scenarios,which is comparable with that in acd1-20,but much more severe than that in nye1-1.Notably,various Chl binding proteins,especially trimeric LHCP Ⅱ,were markedly retained in crn1-1 four days after dark-treatment,possibly due to a lesion in disassociation of protein-pigment complex.Nevertheless,the photochemical efficiency of PSII in crn1-1 declined,even more rapidly,two days after dark-treatment,compared to those in Col-0 and nye1-1.Our results suggest that CRN1 plays a crucial role in Chl degradation,and that loss of its function produces various side-effects,including those on the breakdown of Ch-protein complex and the maintenance of the residual photosynthetic capability during leaf senescence.     

A Label-free Quantitative Proteomics Strategy to Identify E3 Ubiquitin Ligase Substrates Targeted to Proteasome Degradation

The ubiquitin-proteasome system is a central mechanism for controlled proteolysis that regulates numerous cellular processes in eukaryotes. As such, defects in this system can contribute to disease pathogenesis. In this pathway, E3 ubiquitin ligases provide platforms for binding specific substrates, thereby coordinating their ubiquitylation and subsequent degradation by the proteasome. Despite the identification of many E3 ubiquitin ligases, the identities of their specific substrates are still largely unresolved. The ankyrin repeat-containing protein with a suppressor of cytokine signaling box 2 (ASB2) gene that we initially identified as a retinoic acid-response gene in acute promyelocytic leukemia cells encodes the specificity subunit of an E3 ubiquitin ligase complex that is involved in hematopoietic cell differentiation. We have recently identified filamin A and filamin B as the first ASB2 targets and shown that ASB2 triggers ubiquitylation and proteasome-mediated degradation of these proteins. Here a global quantitative proteomics strategy is provided to identify substrates of E3 ubiquitin ligases targeted to proteasomal degradation. Indeed we used label-free methods for quantifying proteins identified by shotgun proteomics in extracts of cells expressing wild-type ASB2 or an E3 ubiquitin ligase-defective mutant of ASB2 under the control of an inducible promoter. Measurements of spectral count and mass spectrometric signal intensity demonstrated a drastic decrease of filamin A and filamin B in myeloid leukemia cells expressing wild-type ASB2 compared with cells expressing an E3 ubiquitin ligase-defective mutant of ASB2. Altogether we provide an original strategy that enables identification of E3 ubiquitin ligase substrates that have to be degraded.The ubiquitin-proteasome system (UPS)¹ plays an essential role in the regulation of protein stability in eukaryotic cells. Degradation of a protein by the UPS entails two successive steps: the covalent attachment of multiple ubiquitin molecules to the protein substrate and its degradation by the 26 S proteasome (1, 2). Ubiquitylation of protein substrates occurs through the sequential action of distinct enzymes: a ubiquitin-activating enzyme, E1; a ubiquitin-conjugating enzyme, E2; and a ubiquitin ligase, E3, responsible for the specific recognition of substrates. Increasing attention has been recently given to the UPS leading to the identification of hundreds of E3 ubiquitin ligases (E3s). Two major classes of E3s have been described: (i) E3s of the HECT (homologous to the E6-associated protein carboxyl terminus) domain family that function as ubiquitin carriers (3, 4) and (ii) E3s of the RING (really interesting new gene) or of the U box families that have no inherent catalytic activity but recruit an E2 enzyme toward substrates (5–7).Classical approaches to identify substrates of E3s are based on the identification of interacting proteins. Although these have successfully led to the identification of a number of substrates of monomeric E3s, identification of substrates of multimeric E3s is very challenging because of the weak affinity of substrates for their requisite specificity subunit and because of the labile nature of the substrate complexed with the specificity subunit (8).Acute promyelocytic leukemia (APL) is associated with six reciprocal translocations always involving the retinoic acid receptor α (RARα) gene (9–11). The RARα protein is a member of the nuclear receptor superfamily that stimulates myeloid differentiation in the presence of its ligand, all-trans-retinoic acid (RA). In more than 95% of APL, the t(15;17) translocation between the promyelocytic leukemia (PML) gene on chromosome 15 and the RARα gene on chromosome 17 produces the PML-RARα fusion protein (12). The PML-RARα protein enhances the repression of RARα target genes by increasing associations with corepressors (13–15) and by recruiting DNA methyltransferases (16). These complexes dissociate from the PML-RARα fusion protein in the presence of pharmacological concentrations of RA perhaps explaining why APL cells are sensitive to RA treatment. Indeed at pharmacological concentrations, RA induces complete remission in a high percentage of APL patients (17–19). By studying RA-induced differentiation of APL cells we have attempted to identify some of the genes that may be up-regulated during this process to further understand the control of growth and differentiation in leukemia (20). One gene identified in this manner, ASB2 (ankyrin repeat-containing protein with a suppressor of cytokine signaling box 2) is an RA-response gene involved in induced differentiation of myeloid leukemia cells (21–23).The ASB2 protein is a subunit of a multimeric E3 ubiquitin ligase of the cullin-RING ligase family (24, 25). The ASB2 suppressor of cytokine signaling box can be divided into a BC box that defines a binding site for the Elongin BC complex and a Cul5 box that determines the binding specificity for Cullin5 (24, 26). Indeed the ASB2 protein, by interacting with the Elongin BC complex, can assemble with a Cullin5/Rbx1 or -2 module to reconstitute an active E3 ubiquitin ligase complex (23–25). Within this complex, the ASB2 protein is the specificity subunit involved in the recruitment of specific substrate(s). Furthermore endogenous ASB2 protein was copurified with ubiquitin ligase activity in RA-treated APL cells suggesting that, during induced differentiation of leukemia cells, the ASB2 protein may target proteins involved in blocking differentiation to destruction by the proteasome machinery (24). We recently identified actin-binding proteins filamin A (FLNa) and filamin B (FLNb) as ASB2 targets and showed that ASB2 triggers ubiquitylation and drives proteasome-mediated degradation of these proteins during RA-induced differentiation of myeloid leukemia cells (23).With the aim to develop a strategy to identify E3 substrates that are degraded by the proteasome, we used an MS approach to identify ASB2 substrates in physiologically relevant settings. Indeed we used label-free quantitative proteomics to identify proteins that are absent or less abundant in cells that express wild-type ASB2 but that accumulate in cells expressing an ASB2 E3 ligase-defective mutant. Application of label-free MS methods that have the advantage to be simple, fast, and cheap enabled the identification of FLNa and FLNb as ASB2 substrates. This study provides a new strategy for the identification of E3 substrates that have to be degraded.