Degradation Of Certain Proteins Research Articles

Ligation to Scavenging Strategy Enables On-Demand Termination of Targeted Protein Degradation.

Event-driven bifunctional molecules, typified by proteolysis targeting chimera (PROTAC) technology, have been successfully applied in degrading many proteins of interest (POI). Due to the unique catalytic mechanism, PROTACs will induce multiple cycles of degradation until the elimination of the target protein. Here, we propose a versatile "Ligation to scavenging" approach to terminate event-driven degradation for the first time. Ligation to the scavenging system consists of a TCO-modified dendrimer (PAMAM-G5-TCO) and tetrazine-modified PROTACs (Tz-PROTACs). PAMAM-G5-TCO can rapidly scavenge intracellular free PROTACs via an inverse electron demand Diels-Alder reaction and terminate the degradation of certain proteins in living cells. Thus, this work proposes a flexible chemical knockdown approach to adjust the levels of POI on-demand in living cells, which paves the way for controlled target protein degradation.

Docosahexaenoic acid inhibits zymogen activation by suppressing vacuolar ATPase activation in cerulein-stimulated pancreatic acinar cells

BackgroundThe premature activation of digestive enzyme zymogens within pancreatic acinar cells is an important early feature of acute pancreatitis. Supraphysiological concentrations of cholecystokinin (CCK) cause intrapancreatic zymogen activation and acute pancreatitis. Stimulation of vacuolar ATPase (vATPase) activity is required for zymogen activation in pancreatic acinar cells. Parkin, a multiprotein E3 ubiquitin ligase complex, promotes vATPase ubiquitination and degradation, which inhibits vATPase activity. Docosahexaenoic acid (DHA), an omega-3 fatty acid, exerts anti-inflammatory effects. It is reported to bind to G-protein coupled receptor 120 (GPR120) and GPR40. DHA induces the degradation of certain proteins by activating ubiquitin-proteasome system in various cells. This study aimed to investigate whether DHA induces Parkin and inhibits vATPase activity, resulting in zymogen inactivation in pancreatic acinar AR42J cells stimulated with cerulein, a CCK analog.ResultsCerulein induced the translocation of the cytosolic V1 domain (E subunit) of vATPase to the membrane, which indicated vATPase activation, and zymogen activation in AR42J cells. DHA suppressed the association of the vATPase with membranes, and zymogen activation (increased trypsin activity and amylase release) induced by cerulein. Pretreatment with a GPR120 antagonist AH-7614, a GPR40 antagonist DC260126, or an ubiquitination inhibitor PYR-41 reduced the effect of DHA on cerulein-induced zymogen activation. Treatment with PYR-41 reversed the DHA-induced decrease in vATPase activation in cerulein-treated cells. Furthermore, DHA increased the level of Parkin in membranes of cerulein-treated cells.ConclusionsDHA upregulates Parkin which inhibits vATPase-mediated zymogen activation, via GPR120 and GPR40, in cerulein-stimulated pancreatic acinar cells.

Abstract C131: HSP27 is necessary in cells showing oncogene hyper-activation.

Abstract The small heat shock protein of 27 KDa (HSP27) encoded by the HSPB1 gene, is a molecular chaperone with anti-aggregation property, it participates in sequestering damaged proteins, and is involved in the proteasomal degradation of certain proteins under stress conditions. Besides its role in stressed condition, HSP27 plays crucial roles within the cell under unstressed conditions and it provides cytoskeletal structural stability and exerts important anti-apoptotic function by binding apoptotic proteins. Clinically, Hsp27 is highly expressed in many cancers including breast, ovarian and prostate carcinomas and is associated with aggressive tumor behaviour, metastasis, poor prognosis and resistance to chemotherapeutics. Using LV driven expression of HSP27 specific shRNA, HSP27 suppression in three gastric cancer cell lines (GTL-16, MKN-45 and EBC-1) showing addiction to MET because of MET gene amplification was able to induce cell death. Moreover, HSP27 silencing synergized in inducing apoptosis with the MET inhibitor JNJ-38877605, which alone was only able to induce cell cycle blockade. Similarly, (H3255) lung carcinoma and (DiFi) colorectal carcinoma cell lines which carry EGFR amplification and are susceptible to EGFR inhibition were exquisitely susceptible to HSP27 silencing, that induced cell death. HSP27 silenced in twenty human colorectal (CRC) cell lines with either wt KRAS or harbouring KRAS mutations (SW620, LS513, LS1034) or BRAF mutations (OXCO-1, COLO741, COLO-201, COLO-205), Among them the LS513 and LS1034 cells and the COLO-201 and COLO-205, addicted to KRAS and BRAF, respectively, came out to be also addicted to HSP27, as they undergo apoptosis by HSP27 silencing. Taken together, these data suggest that cells addicted to oncogene over-activation require HSP27 for survival and that HSP27 interferes with the effectiveness of targeted agents. Due to its involvement in cancer development, the inhibition of HSP27 has been proposed as a potential cancer treatment strategy. Antisense and peptide aptamer strategies have shown that targeting HSP27 increases cancer cell death in vitro and in vivo in preclinical models. More important, an antisense drug inhibiting HSP27 is available for human therapy and is ongoing Phase II clinical trials. Therefore, HSP27 knockdown in combination with targeted therapies can be envisaged as a viable therapeutic approach for clinical application. Citation Information: Mol Cancer Ther 2013;12(11 Suppl):C131. Citation Format: Johndavid Konda, Martina Olivero, Daniele Musiani, Simona Lamba, Alberto Bardelli, Federica Di Nicolantonio, Maria Flavia Di Renzo. HSP27 is necessary in cells showing oncogene hyper-activation. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2013 Oct 19-23; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2013;12(11 Suppl):Abstract nr C131.

Insight into the Intermolecular Recognition Mechanism between Keap1 and IKKβ Combining Homology Modelling, Protein-Protein Docking, Molecular Dynamics Simulations and Virtual Alanine Mutation

Degradation of certain proteins through the ubiquitin-proteasome pathway is a common strategy taken by the key modulators responsible for stress responses. Kelch-like ECH-associated protein-1(Keap1), a substrate adaptor component of the Cullin3 (Cul3)-based ubiquitin E3 ligase complex, mediates the ubiquitination of two key modulators, NF-E2-related factor 2 (Nrf2) and IκB kinase β (IKKβ), which are involved in the redox control of gene transcription. However, compared to the Keap1-Nrf2 protein-protein interaction (PPI), the intermolecular recognition mechanism of Keap1 and IKKβ has been poorly investigated. In order to explore the binding pattern between Keap1 and IKKβ, the PPI model of Keap1 and IKKβ was investigated. The structure of human IKKβ was constructed by means of the homology modeling method and using reported crystal structure of Xenopus laevis IKKβ as the template. A protein-protein docking method was applied to develop the Keap1-IKKβ complex model. After the refinement and visual analysis of docked proteins, the chosen pose was further optimized through molecular dynamics simulations. The resulting structure was utilized to conduct the virtual alanine mutation for the exploration of hot-spots significant for the intermolecular interaction. Overall, our results provided structural insights into the PPI model of Keap1-IKKβ and suggest that the substrate specificity of Keap1 depend on the interaction with the key tyrosines, namely Tyr525, Tyr574 and Tyr334. The study presented in the current project may be useful to design molecules that selectively modulate Keap1. The selective recognition mechanism of Keap1 with IKKβ or Nrf2 will be helpful to further know the crosstalk between NF-κB and Nrf2 signaling.

Selective Sorting of Cargo Proteins into Bacterial Membrane Vesicles

In contrast to the well established multiple cellular roles of membrane vesicles in eukaryotic cell biology, outer membrane vesicles (OMV) produced via blebbing of prokaryotic membranes have frequently been regarded as cell debris or microscopy artifacts. Increasingly, however, bacterial membrane vesicles are thought to play a role in microbial virulence, although it remains to be determined whether OMV result from a directed process or from passive disintegration of the outer membrane. Here we establish that the human oral pathogen Porphyromonas gingivalis has a mechanism to selectively sort proteins into OMV, resulting in the preferential packaging of virulence factors into OMV and the exclusion of abundant outer membrane proteins from the protein cargo. Furthermore, we show a critical role for lipopolysaccharide in directing this sorting mechanism. The existence of a process to package specific virulence factors into OMV may significantly alter our current understanding of host-pathogen interactions.

Heat Shock Proteins: Cell Protection through Protein Triage

Heat shock proteins (HSPs) are chaperones that catalyze the proper folding of nascent proteins and the refolding of denatured proteins. The ubiquitin-proteasome system is an error-checking system that directs improperly folded proteins for destruction. A coordinated interaction between the HSPs (renaturation) and the proteasome (degradation) must exist to assure protein quality control mechanisms. Although it still remains unknown how the decision of folding vs. degradation is taken, many pieces of evidence demonstrate that HSPs interact directly or indirectly with the proteasome, assuring quite selectively the proteasomal degradation of certain proteins under stress conditions. In this review, we will describe the different data that demonstrate a role for HSP90, HSP70, HSP27, and alpha-B-crystallin in the partitioning of proteins to either one of these pathways, referred as protein triage.

Host cell sumoylation level influences papillomavirus E2 protein stability

The stability of papillomavirus E2 proteins is regulated by proteasomal degradation, and regulation of degradation could contribute to the higher expression levels of E2 proteins observed in suprabasal layers of differentiated skin. We have recently shown that the E2 proteins are modified by sumoylation [Wu Y-C, Roark AA, Bian X-L, Wilson, VG (2008) Virol 378:329–338], and that sumoylation levels are up-regulated during keratinocyte differentiation [Deyrieux AF, Rosas-Acosta G, Ozbun MA, Wilson VG (2007) J Cell Sci 120:125–136]. These observations, coupled with the known ability of sumoylation to prevent proteasomal degradation of certain proteins, suggested that this modification might contribute to stabilizing E2 proteins in suprabasal keratinocytes. Conditions that increased overall sumoylation were found to increase the intracellular amounts of the HPV11, 16, and 18 E2 proteins. No effect of sumoylation was seen on E2 transcripts, and the increased levels of E2 proteins resulted from a greatly increased half-life for the E2 proteins. In vitro studies confirmed that sumoylation could block the proteasomal degradation of the 16E2 protein. Interestingly, this stabilization effect was indirect as it did not require sumoylation of 16E2 itself and must be acting through sumoylation of a cellular target(s). This sumoylation-dependent, indirect stabilization of E2 proteins is a novel process that may couple E2 levels to changes in the cellular environment. Specifically, our results suggest that the levels of papillomavirus E2 protein could be up-regulated in differentiating keratinocytes in response to the increased overall sumoylation that accompanies differentiation.

59. rAAV-Mediated Nigral Parkin Over-Expression Is Neuroprotective in the 6-OHDA Rat Model of Parkinson's Disease

Parkinson's disease (PD) is a neuro-degenerative disease characterized by the progressive loss of dopaminergic neurons in the substantia nigra pars compacta (SNc). Although the underlying cause of the idiopathic form of the disease has yet to be found, several genes have been found that cause familial forms of PD. One such gene is parkin which has been identified as an ubiquitin E3 ligase which is involved in the ubiquitination and subsequent degradation of certain proteins. Mutations in this gene leads to an early onset form of PD called Autosomal Recessive Juvenile Parkinsonism (ARJP). Given the role of parkin in protein clearance, and the presence of Lewy Bodies in the disease, it is possible that protein aggregation is a neurotoxic event in idiopathic PD, and as such, over-expression of parkin may be neuroprotective in animal models of PD by enhancing the degradation of damaged or misfolded proteins. This was tested by doing intranigral injections of a recombinant adeno-associated virus (rAAV) expressing either a human form of parkin or green fluorescent protein (GFP) as control for viral transduction. 4 weeks following the viral injections all animals received a 4 site intrastriatal injection of the neurotoxin 6-hydroxydopamine (6-OHDA) resulting in a near complete depletion of striatal and nigral dopamine. Behavioral evaluation following the lesion indicates that parkin confers partial protection of the nigrostriatal tract. Amphetaine-induced rotational behavior was significantly reduced in the treatment group (|[minus]|67% as compared to controls). Testing for spontaneous limb-use in the cylinder testing paradigm also showed a slight, but significant improvement, where parkin treated animals used their affected limb approximately 20% of the time versus 10% for the control group. These data indicate that specific nigral over-expression of human parkin is partially neuroprotective at the functional level against a strong toxic stimulus, intrastriatal 6-OHDA. Additionally, the data suggest that enhanced ubiquitin-mediated clearance of damaged proteins is protective against a non-specific insult in nigrostriatal dopamine neurons.

Biosynthetic incorporation of oxidized amino acids into proteins and their cellular proteolysis

We demonstrate that oxidized amino acids can be incorporated into proteins by protein synthesis. The level of incorporation into protein was dependent on the concentration of oxidized amino acid supplied to the cells. At low levels of incorporation, the oxidized amino acids examined increased the degradation rate of the cell proteins. Degradation of certain proteins containing high levels of DOPA (but not ortho or meta tyrosine) was decreased to below the basal degradation rates suggesting that DOPA may contribute to proteins becoming resistant to proteolysis. Changes in the degradation rates of the oxidized amino acid-containing proteins was shown to have no impact on the degradation rates of native proteins, indicating that the activity of the degradative machinery was not affected. We demonstrate that oxidized proteins are selectively degraded by the proteasomes and provide evidence to suggest that the proteasomes and the endosomal-lysosomal systems may act in sequence as well as in parallel. The incorporation approach, unlike cell studies in which an exogenous oxidant is used, allows the degradation rates of the oxidatively modified proteins to be selectively measured, offering a greater sensitivity as well as greatly reducing toxicity to the cell and avoiding oxidative modification of other cell components.

Distinct cleavage specificity of human cathepsin E at neutral pH with special preference for Arg-Arg bonds.

In order to clarify the potential role of cathepsin E at neutral pH, the cleavage specificity of human cathepsin E was examined at pH 7.4 toward reduced-carboxymethylated(RCm-)ribonuclease A and various bioactive and related peptides. The specificity of the enzyme at pH 7.4 was found to be considerably different from that at acidic pH; preferential cleavages were observed with Arg-X and Glu-X bonds, which are not the major cleavage sites at acidic pH. Moreover, the Arg-Arg bond was found to be the most preferential site of cleavage. This unique specificity observed at pH 7.4 suggests the possibility that cathepsin E might be involved in processing and/or degradation of certain proteins and/or peptides at or near neutral pH in vivo.

Interference with gene expression induces rapid apoptosis in p53-null T lymphoma cells.

Two p53-null T lymphoma cell lines proved to be highly sensitive to inhibition of gene expression. With either actinomycin D or cycloheximide, apoptosis commenced within 2 h, as indicated by loss of membrane integrity, degradation of certain proteins (including the phosphatase calcineurin) and DNA fragmentation. These effects were ablated by co-expression of Bcl-2 or co-incubation with the caspase inhibitor Z-VAD-fmk. These results suggest that the apoptotic machinery is in place in these cells but held in check by an unknown labile protein, which probably acts upstream of Bcl-2. Although cycloheximide can activate the JNK or p38 MAP kinases in some cells, neither was implicated here. However, disruption of phosphoinositide 3-kinase signaling may be involved, because the cells were also sensitive to wortmannin. The high sensitivity of the p53-null lymphoma cells to inhibitors of gene expression suggests that such inhibitors might prove useful in the cytotoxic therapy of certain tumors.

The calpain–calpastatin system and protein degradation in fusing myoblasts

Calpain (Ca 2+-activated cysteine protease) induced proteolysis has been suggested to play a role in myoblast fusion. We previously found that calpastatin (the endogenous inhibitor of calpain) diminishes markedly in myoblasts during myoblast differentiation just prior to the start of fusion, allowing Ca 2+-induced calpain activation at that stage. Here, we show that a limited degradation of some proteins occurs within the myoblasts undergoing fusion, but not in proliferating myoblasts. The protein degradation is observed at the stage when calpastatin is low. Protein degradation within the myoblasts and myoblast fusion are inhibited by EGTA, by the cysteine protease inhibitors calpeptin and E-64d and by calpastatin. The degradation appears to be selective for certain myoblast proteins. Integrin β1 subunit, talin and β-tropomyosin are degraded in the fusing myoblasts, whereas α-actinin, β-tubulin and α-tropomyosin are not. A similar pattern of degradation is observed in lysates of proliferating myoblasts when Ca 2+ and excess calpain are added, a degradation that is inhibited by calpastatin. The results support the notion that degradation of certain proteins is required for myoblast fusion and that calpain participates in the fusion-associated protein degradation. Participation of calpain is made possible by a change in calpain/calpastatin ratio, i.e., by a diminution in calpastatin level from a high level in the proliferating myoblasts to a low level in the differentiating myoblasts. Degradation of certain proteins, known to be responsible for the stability of the membrane–skeleton organization and for the interaction of the cell with the extracellular matrix, would allow destabilization of the membrane and the creation of membrane fusion-potent regions.

Proteasome Inhibition Leads to a Heat-shock Response, Induction of Endoplasmic Reticulum Chaperones, and Thermotolerance

The accumulation of misfolded proteins in the cytosol leads to increased expression of heat-shock proteins, while accumulation of such proteins in the endoplasmic reticulum (ER) stimulates the expression of many ER resident proteins, most of which function as molecular chaperones. Recently, inhibitors of the proteasome have been identified that can block the rapid degradation of abnormal cytosolic and ER-associated proteins. We therefore tested whether these agents, by causing the accumulation of abnormal proteins, might stimulate the expression of cytosolic heat-shock proteins and/or ER molecular chaperones and thereby induce thermotolerance. Exposure of Madin-Darby canine kidney cells to various proteasome inhibitors, including the peptide aldehydes (MG132, MG115, N-acetyl-leucyl-leucyl-norleucinal) and lactacystin, inhibited the degradation of short-lived proteins and increased markedly the levels of mRNAs encoding cytosolic heat-shock proteins (Hsp70, polyubiquitin) and ER chaperones (BiP, Grp94, ERp72), as shown by Northern blot analysis. However, inhibitors of cysteine proteases (E64), serine proteases (leupeptin), or metalloproteases (1, 10-phenanthroline) had no effect on the levels of these mRNAs. The relative efficacies of the peptide aldehyde inhibitors in inducing these mRNAs correlated with their potencies against the proteasome. Furthermore, reduction of the aldehyde group of MG132 decreased its inhibitory effect on proteolysis and largely prevented the induction of these mRNAs. Although treatment with the proteasome inhibitors caused rapid increases in mRNA levels (as early as 2 h after treatment with MG132), the inhibitors did not detectably affect total protein synthesis, total protein secretion, ER morphology, or the retention of ER-lumenal proteins, even after 18 h of treatment. Together, the findings suggest that inhibition of proteasome function induces heat-shock proteins and ER chaperones due to the accumulation of sufficient amounts of abnormal proteins and/or the inhibition of degradation of a key regulatory factor (e.g. heat-shock factor). Since expression of heat-shock proteins can protect cells from thermal injury, we tested whether the proteasome inhibitors might also confer thermotolerance. Treatment of cells with MG132 for as little as 2 h, markedly increased the survival of cells subjected to high temperatures (up to 46 degrees C). Thus, these agents may have applications in protecting against cell injury.

Protein quality control: triage by chaperones and proteases.

Proteases and chaperones together serve to maintain quahty control of cellular proteins. Both types of en­ zymes have as their substrates the variety of misfolded and partially folded proteins that arise from slow rates of folding or assembly, chemical or thermal stress, intrinsic structural instability, and biosynthetic errors. The pri­ mary function of classical chaperones, such as the Esch­ erichia coh DnaK/Hsp70 and its cochaperones, DnaJ and GrpE, and GroEL/Hsp60 and its cochaperone, GroES, is to modulate protein folding pathways, thereby prevent­ ing misfolding and aggregation, promoting refolding and proper assembly. Recent work has demonstrated that ATP-dependent proteases, as well as closely related pro­ teins, have intrinsic chaperone activity, suggesting that the initial steps in energy-dependent protein degradation may be similar to those of chaperone-dependent protein folding. The classical chaperones are also required for degradation of certain proteins in vivo, but we propose below that generally they affect proteolysis indirectly by maintaining proteins in soluble forms that would other­ wise aggregate and become inaccessible to proteases. In this review we present the evidence linking the activi­ ties of chaperones and proteases, and propose a general model for what can be thought of as a triage system for handling misfolded proteins in vivo, assuring swift re­ folding of proteins with functional potential and rapid degradation of irreversibly denatured or damaged pro­ teins.

Involvement of the molecular chaperone Ydj1 in the ubiquitin-dependent degradation of short-lived and abnormal proteins in Saccharomyces cerevisiae.

In Escherichia coli and mitochondria, the molecular chaperone DnaJ is required not only for protein folding but also for selective degradation of certain abnormal polypeptides. Here we demonstrate that in the yeast cytosol, the homologous chaperone Ydj1 is also required for ubiquitin-dependent degradation of certain abnormal proteins. The temperature-sensitive ydj1-151 mutant showed a large defect in the overall breakdown of short-lived cell proteins and abnormal polypeptides containing amino acid analogs, especially at 38 degrees C. By contrast, the degradation of long-lived cell proteins, which is independent of ubiquitin, was not altered nor was cell growth affected. The inactivation of Ydj1 markedly reduced the rapid, ubiquitin-dependent breakdown of certain beta-galactosidase (beta-gal) fusion polypeptides. Although degradation of N-end rule substrates (arginine-beta-gal and leucine-beta-gal) and the B-type cyclin Clb5-beta-gal occurred normally, degradation of the abnormal polypeptide ubiquitin-proline-beta-gal (Ub-P-beta-gal) and that of the short-lived normal protein Gcn4 were inhibited. As a consequence of reduced degradation of Ub-P-beta-gal, the beta-gal activity was four to five times higher in temperature-sensitive ydj1-151 mutant cells than in wild-type cells; thus, the folding and assembly of this enzyme do not require Ydj1 function. In wild-type cells, but not in ydj1-151 mutant cells, this chaperone is associated with the short-lived substrate Ub-P-beta-gal and not with stable beta-gal constructs. Furthermore, in the ydj1-151 mutant, the ubiquitination of Ub-P-beta-gal was blocked and the total level of ubiquitinated protein in the cell was reduced. Thus, Ydj1 is essential for the ubiquitin-dependent degradation of certain proteins. This chaperone may facilitate the recognition of unfolded proteins or serve as a cofactor for certain ubiquitin-ligating enzymes.

Rapid degradation of an abnormal protein in Escherichia coli involves the chaperones GroEL and GroES.

In Escherichia coli, the molecular chaperones (DnaK, DnaJ, and GrpE) are essential for the rapid degradation of certain proteins. To see if chaperones are involved more generally in proteolysis, we studied the degradation of a short-lived fusion protein, CRAG, which associates with DnaK and GroEL in vivo. Its rapid degradation requires ATP and ClpP, the proteolytic subunit of protease Ti (Clp). However, this process is not reduced in strains lacking the complementary ATPase subunit, ClpA, or its homologs, ClpB and ClpX. At 37 degrees C, but not at 42 degrees C, protease La also contributes partially to CRAG degradation. Nevertheless, CRAG is not degraded in cell-free extracts or upon incubation with ClpP or protease La. We tested whether the chaperones associated with CRAG might be involved in its degradation. CRAG breakdown was accelerated 2-3-fold in strains with high levels of heat-shock proteins (hsps), i.e. in those that overproduce the hsp transcription factor (sigma 32) or carry a dnaK deletion. A similar stimulation of proteolysis was observed in cells overproducing GroEL or both GroEL and GroES; in these cells, more CRAG was associated with GroEL than in the wild type. In a temperature-sensitive groEL44 mutant at the nonpermissive temperature, CRAG breakdown was accelerated, and more CRAG was found complexed with GroEL. However, in a temperature-sensitive groES mutant, CRAG was completely stable at the nonpermissive temperature and accumulated bound to GroEL. These findings indicate that the association of CRAG with GroEL is a rate-limiting step in CRAG degradation, which also requires a subsequent action of GroES. We propose that if the hsp60/hsp10 chaperonins fail to catalyze the proper folding of a protein, they can facilitate its rapid degradation.

Involvement of intracellular calcium in protein secretion in Dictyostelium discoideum.

We reported previously that Ca2+ depletion of Dictyostelium discoideum cells severely inhibits extracellular cyclic nucleotide phosphodiesterase (PD) synthesis at a post-transcriptional step. In this study, further experiments were performed to learn more about the nature of this phenomenon. Examination of the polysomal distribution of PD transcripts in control cells and in cells depleted of Ca2+ by incubation with EGTA and A23187 (EA) suggested that inhibition of PD production does not involve translational control. Kinetic analysis of this inhibitory process revealed that soluble, intracellular PD activity, synthesized from either the 2.4 or 1.9 kb PD mRNA, decreased very rapidly upon addition of EA. Furthermore, this decrease in activity was accompanied by the preferential loss of PD-related polypeptides, indicating a proteolytic event. EA-induced PD degradation required cellular energy and concomitant protein synthesis but was unaffected by most of the lysosomotropic agents tested. Therefore, PD proteolysis might not occur in the lysosome. In cell fractionation experiments, the EA-sensitive, intracellular PD activity comigrated with a rough ER marker in Percoll/KCl gradients. In addition to its effect on the PD, EA were also observed to inhibit production and rapidly lower the intracellular levels of another secreted glycoprotein, the PD inhibitor. Together, these results suggest that depletion of some intracellular Ca2+ store(s) in Dictyostelium, possibly the ER, disrupts the normal function of the secretory pathway, resulting in selective degradation of certain proteins.

5’-Diphosphate-guanosine-3’-diphosphate (ppGpp) Stimulates the Degradation of Certain Proteins by ATP-dependent Protease La from Escherichia coli

5’-Diphosphate-guanosine-3’-diphosphate (ppGpp) Stimulates the Degradation of Certain Proteins by ATP-dependent Protease La from Escherichia coli

UBC1 encodes a novel member of an essential subfamily of yeast ubiquitin-conjugating enzymes involved in protein degradation.

The covalent attachment of ubiquitin to cellular proteins is catalyzed by members of a family of ubiquitin-conjugating enzymes. These enzymes participate in a variety of cellular processes, including selective protein degradation, DNA repair, cell cycle control, and sporulation. In the yeast Saccharomyces cerevisiae, two closely related ubiquitin-conjugating enzymes, UBC4 and UBC5, have recently been shown to mediate the selective degradation of short-lived and abnormal proteins. We have now identified a third distinct member of this class of ubiquitin-conjugating enzymes, UBC1. UBC1, UBC4 and UBC5 are functionally overlapping and constitute an enzyme family essential for cell growth and viability. All three mediate selective protein degradation, however, UBC1 appears to function primarily in the early stages of growth after germination of spores. ubc1 mutants generated by gene disruption display only a moderate slow growth phenotype, but are markedly impaired in growth following germination. Moreover, yeast carrying the ubc1ubc4 double mutation are viable as mitotic cells, however, these cells fail to survive after undergoing sporulation and germination. This specific requirement for UBC1 after a state of quiescence suggests that degradation of certain proteins may be crucial at this transition point in the yeast life cycle.