GrpE-like regulation of the hsc70 chaperone by the anti-apoptotic protein BAG-1.

The BAG-1 protein appears to inhibit cell death by binding to Bcl-2, the Raf-1 protein kinase, and certain growth factor receptors, but the mechanism of inhibition remains enigmatic. BAG-1 also interacts with several steroid hormone receptors which require the molecular chaperones Hsc70 and Hsp90 for activation. Here we show that BAG-1 is a regulator of the Hsc70 chaperone. BAG-1 binds to the ATPase domain of Hsc70 and, in cooperation with Hsp40, stimulates Hsc70's steady-state ATP hydrolysis activity approximately 40-fold. Similar to the action of the GrpE protein on bacterial Hsp70, BAG-1 accelerates the release of ADP from Hsc70. Thus, BAG-1 regulates the Hsc70 ATPase in a manner contrary to the Hsc70-interacting protein Hip, which stabilizes the ADP-bound state. Intriguingly, BAG-1 and Hip compete in binding to the ATPase domain of Hsc70. Our results reveal an unexpected diversity in the regulation of Hsc70 and raise the possibility that the observed anti-apoptotic function of BAG-1 may be exerted through a modulation of the chaperone activity of Hsc70 on specific protein folding and maturation pathways.     

BAG-1 associates with Hsc70.Tau complex and regulates the proteasomal degradation of Tau protein

Intraneuronal accumulation of phosphorylated Tau protein is a molecular pathology found in many forms of dementia, including Alzheimer disease. Research into possible mechanisms leading to the accumulation of modified Tau protein and the possibility of removing Tau protein from the system have revealed that the chaperone protein system can interact with Tau and mediate its degradation. Hsp70/Hsc70, a member of the chaperone protein family, interacts with Tau protein and mediates proper folding of Tau and can promote degradation of Tau protein under certain circumstances. However, because Hsp70/Hsc70 has many binding partners that can mediate its activity, there is still much to discover about how Hsp70 acts in vivo to regulate Tau protein. BAG-1, an Hsp70/Hsc70 binding partner, has been implicated as a mediator of neuronal function. In this work we show that BAG-1 associates with Tau protein in an Hsc70-dependent manner. Overexpression of BAG-1 induced an increase in Tau levels, which is shown to be due to an inhibition of protein degradation. We further show that BAG-1 can inhibit the degradation of Tau protein by the 20 S proteasome but does not affect the ubiquitination of Tau protein. RNA-mediated interference depletion of BAG-1 leads to a decrease in total Tau protein levels as well as promoting hyperphosphorylation of the remaining protein. Induction of Hsp70 by heat shock enhanced the increase of Tau levels in cells overexpressing BAG-1 but induced a decrease of Tau levels in cells that were depleted of BAG-1. Finally, BAG-1 is highly expressed in neurons bearing Tau tangles in a mouse model of Alzheimer disease. This data suggests a molecular mechanism through which Tau protein levels are regulated in the cell and possible consequences for the pathology and treatment of Alzheimer disease.     

Cooperation of a ubiquitin domain protein and an E3 ubiquitin ligase during chaperone/proteasome coupling.

BACKGROUND: Molecular chaperones recognize nonnative proteins and orchestrate cellular folding processes in conjunction with regulatory cofactors. However, not every attempt to fold a protein is successful, and misfolded proteins can be directed to the cellular degradation machinery for destruction. Molecular mechanisms underlying the cooperation of molecular chaperones with the degradation machinery remain largely enigmatic so far. RESULTS: By characterizing the chaperone cofactors BAG-1 and CHIP, we gained insight into the cooperation of the molecular chaperones Hsc70 and Hsp70 with the ubiquitin/proteasome system, a major system for protein degradation in eukaryotic cells. The cofactor CHIP acts as a ubiquitin ligase in the ubiquitination of chaperone substrates such as the raf-1 protein kinase and the glucocorticoid hormone receptor. During targeting of signaling molecules to the proteasome, CHIP may cooperate with BAG-1, a ubiquitin domain protein previously shown to act as a coupling factor between Hsc/Hsp70 and the proteasome. BAG-1 directly interacts with CHIP; it accepts substrates from Hsc/Hsp70 and presents associated proteins to the CHIP ubiquitin conjugation machinery. Consequently, BAG-1 promotes CHIP-induced degradation of the glucocorticoid hormone receptor in vivo. CONCLUSIONS: The ubiquitin domain protein BAG-1 and the CHIP ubiquitin ligase can cooperate to shift the activity of the Hsc/Hsp70 chaperone system from protein folding to degradation. The chaperone cofactors thus act as key regulators to influence protein quality control.     

Involvement of a chaperone regulator, Bcl2-associated athanogene-4, in apolipoprotein B mRNA editing

Apobec-1 is the catalytic subunit of a multicomponent editosome complex that mediates apolipoprotein B (apoB) mRNA editing. We isolated a novel apobec-1-interacting protein by yeast two-hybrid cloning and identified the protein as BAG-4. BAG-4, a chaperone-regulating protein, also known as SODD (silencer of death domains), is a member of the BAG family of proteins. In this report, we found that apobec-1 is localized in the perinucleolar compartment in HepG2 cells and rat liver MCR-RH7777 cells. BAG-4 binds to apobec-1 via its N-terminal region independent of the BAG domain. It is ubiquitously expressed with predominant occurrence in human pancreas, heart, brain, and placenta. Immunoprecipitation experiments confirmed that BAG-4 interacts with Hsc70/Hsp90 in HepG2 cells. BAG-4 tagged with green fluorescent protein (GFP) or FLAG was localized both in cytoplasm of mouse BNLCL.2 liver cells and human liver hepatoma HepG2 cells. After heat shock, GFP-BAG-4 co-localizes with Hsc70 in the nucleus in HepG2 cells, whereas GFP-BAG-4 mutants lacking the BAG domain remain perinuclear. BAG-4 has no effects on apoB mRNA editing in vitro. However, unlike other apobec-1 complementation factors studied to date, antisense knockdown of BAG-4 in BNLCL.2 cells and in MCR-RH7777 cells increases rather than decreases endogenous apoB mRNA editing. Overexpression of BAG-4 in MCR-RH7777 cells also suppresses apoB mRNA editing. ApoB-48 production also increases with antisense BAG-4 expression in MCR-RH7777 cells. We previously showed that apoB mRNA editing is an intranuclear event (Lau, P. P., Xiong, W. J., Zhu, H. J., Chen, S. H., and Chan, L. (1991) J. Biol. Chem. 266, 20550-20554). Thus, BAG-4 overexpression down-regulates apoB mRNA editing by shuttling apobec-1 from the intranuclear perinucleolar compartment to the cytoplasm. We propose that BAG-4 functions as a negative regulator for apobec-1-mediated apoB mRNA editing through its ability to suppress the Hsp/Hsc70 chaperone activity and thereby editosome formation and, as a consequence, prevents nuclear localization of the apobec-1 editosome.     

p53-inducible human homologue of Drosophila seven in absentia (Siah) inhibits cell growth: suppression by BAG-1.

The Drosophila seven in absentia (sina) gene is required for R7 photoreceptor cell formation during Drosophila eye development, where it functions within the Ras/Raf pathway and targets other proteins for degradation via associations with a ubiquitin-conjugating enzyme. Recently, a mammalian sina homologue was reported to be a p53-inducible gene in a myeloid leukemia cell line. To explore the function of human SINA-homologous (Siah) proteins, expression plasmids encoding Siah-1A were transiently transfected into 293 epithelial cells and GM701 fibroblast cells, resulting in growth arrest without induction of apoptosis. We discovered that BAG-1, a ubiquitin-like Hsp70/Hsc70-regulating protein, is a negative regulator of Siah-1A. Siah-1A was identified as a BAG-1-binding protein via yeast two-hybrid methods. Specific interaction of BAG-1 with Siah-1A was also demonstrated by in vitro binding experiments using glutathione S-transferase fusion proteins and co-immunoprecipitation studies. Siah-1A-induced growth arrest in 293 and GM701 cells was abolished by co-transfection of wild-type BAG-1 with Siah-1A but not by a C-terminal deletion mutant of BAG-1 that fails to bind Siah-1A. Over-expression of BAG-1 significantly inhibited p53-induced growth arrest in 293 cells without preventing p53 transactivation of reporter gene plasmids. BAG-1 also prevented growth arrest following UV-irradiation-induced genotoxic injury without interfering with accumulation of p53 protein or p21(waf-1) expression. BAG-1 functions downstream of p53-induced gene expression to inhibit p53-mediated suppression of cell growth, presumably by suppressing the actions of Siah-1A. We suggest that Siah-1A may be an important mediator of p53-dependent cell-cycle arrest and demonstrate that Siah-1A is directly inhibited by BAG-1.     

The retinoblastoma protein (Rb) as an anti-apoptotic factor: expression of Rb is required for the anti-apoptotic function of BAG-1 protein in colorectal tumour cells

Although the retinoblastoma-susceptibility gene RB1 is inactivated in a wide range of human tumours, in colorectal cancer, the retinoblastoma protein (Rb) function is often preserved and the RB locus even amplified. Importantly, we have previously shown that Rb interacts with the anti-apoptotic Bcl-2 associated athanogene 1 (BAG-1) protein, which is highly expressed in colorectal carcinogenesis. Here we show for the first time that Rb expression is critical for BAG-1 anti-apoptotic activity in colorectal tumour cells. We demonstrate that Rb expression not only increases the nuclear localisation of the anti-apoptotic BAG-1 protein, but that expression of Rb is required for inhibition of apoptosis by BAG-1 both in a γ-irradiated Saos-2 osteosarcoma cell line and colorectal adenoma and carcinoma cell lines. Further, consistent with the fact that nuclear BAG-1 has previously been shown to promote cell survival through increasing nuclear factor (NF)-κB activity, we demonstrate that the ability of BAG-1 to promote NF-κB activity is significantly inhibited by repression of Rb expression. Taken together, data presented suggest a novel function for Rb, promoting cell survival through regulating the function of BAG-1. As BAG-1 is highly expressed in the majority of colorectal tumours, targeting the Rb–BAG-1 complex to promote apoptosis has exciting potential for future therapeutic development.     

BAG-1 proteins protect cardiac myocytes from simulated ischemia/reperfusion-induced apoptosis via an alternate mechanism of cell survival independent of the proteasome

BAG-1 (Bcl-2-associated athanogene-1) proteins interact with the HSC70 and HSP70 heat shock proteins and have been proposed to promote cell survival by coordinating the function of these chaperones with the proteasome to facilitate protein degradation. Consistent with this proposal, previous analyses in cancer cells have demonstrated that BAG-1 requires protein domains important for HSC70/HSP70 and proteasome binding in order to interfere with the growth inhibition induced by heat shock (Townsend, P. A., Cutress, R. I., Sharp, A., Brimmell, M., and Packham, G. (2003) Cancer Res., 63, 4150-4157). Moreover, cellular stress triggered the relocalization of the cytoplasmic BAG-1S (approximately 36 kDa) isoform to the nucleus, and both BAG-1S and the constitutively nuclear localized BAG-1L (approximately 50 kDa) isoform suppressed heat shock-induced apoptosis to the same extent, suggesting a critical role in the nucleus. Because ischemia (I) and reperfusion (R) are important stress signals in acute and chronic heart disease, we have examined the expression and function of BAG-1 proteins in primary cardiac myocytes (CMs) and the Langendorff-perfused intact heart. The expression of both BAG-1 isoforms, BAG-1S and BAG-1L, was rapidly induced following ischemia in rat CM, and this was maintained during subsequent reperfusion. In control hearts, BAG-1S and BAG-1L were readily detectable in both the nucleus and the cytoplasm. However, BAG-1S did not relocate to the nucleus following simulated I/R. BAG-1 interacted with both RAF-1 and HSC70 in CMs and the whole heart, and binding to HSC70 was increased following I/R. Overexpression of the human BAG-1S and BAG-1 M isoforms significantly reduced CM apoptosis following simulated I/R. By contrast, BAG-1L or BAG-1S fused to a heterologous nuclear localization sequence failed to protect CM. Finally, overexpression of BAG-1 deletion and point mutants unable to bind HSC70/HSP70 failed to offer cardioprotection. Surprisingly, a deletion mutant lacking the N-terminal ubiquitin-like domain, which mediates interaction with the proteasome, still promoted cardioprotection. Therefore, BAG-1 has a novel cardioprotective role, mediated via association with HSC70/HSP70, which is critical upon cytoplasmic localization but independent of the BAG-1 ubiquitin-like domain. Our studies demonstrate that BAG-1 can influence cellular response to stress by multiple mechanisms, potentially influenced by the cell type and nature of the stress signal.     

BAG-1, an anti-apoptotic tumour marker

BAG-1 is a multifunctional and anti-apoptotic or anti-cell death protein that interacts with a variety of cellular proteins and affects their functions. On the cell surface, it binds to the cytosolic domain of the growth factor receptors and enhances the protection from cell death triggered by growth factor receptors. In the cytosol, it binds to Bcl-2 and heat shock protein, and modulates their functions. In the nucleus, it binds to a variety of nuclear hormone receptors and inhibits hormone-induced apoptosis. BAG-1 is widely overexpressed in a variety of tumour cell lines and cancer tissues. In addition, differential expression of BAG-1 isoforms has been observed. Preclinical studies indicate that overexpression of BAG-1, especially its nuclear and cytoplasmic isoforms, may be useful as a prognostic and/or predictive biomarker. Pilot clinical studies have demonstrated that overexpression of nuclear BAG-1 may be associated with a shorter survival in breast and laryngeal carcinomas. Conversely, overexpression of cytoplasmic BAG-1 may be associated with a better clinical outcome in early stage breast cancer and in non-small cell lung cancer. Further large-scale clinical studies are warranted to establish the role of BAG-1 as a novel prognostic and/or predictive biomarker in the clinical management of these common malignancies.     

BAG-1 is a novel cytoplasmic binding partner of the membrane form of heparin-binding EGF-like growth factor: a unique role for proHB-EGF in cell survival regulation

Several cell functions related to growth and survival regulation have been attributed specifically to the membrane form of heparin-binding EGF-like growth factor (proHB-EGF), rather than to the diffusible, processed HB-EGF isoform. These findings suggest the existence of a functional binding partner specifically for the membrane form of the growth factor. In this study we have identified the prosurvival cochaperone, BAG-1, as a protein that interacts with the cytoplasmic tail domain of proHB-EGF. Interaction between BAG-1 and the 24-amino acid proHB-EGF cytoplasmic tail was initially identified in a yeast two-hybrid screen and was confirmed in mammalian cells. The proHB-EGF tail bound BAG-1 in an hsp70-independent manner and within a 97-amino acid segment that includes the ubiquitin homology domain in BAG-1 but does not include the hsp70 binding site. Effects of BAG-1 and proHB-EGF co-expression were demonstrated in cell adhesion and cell survival assays and in quantitative assays of regulated secretion of soluble HB-EGF. Because the BAG-1 binding site is not present on the mature, diffusible form of the growth factor, these findings suggest a new mechanism by which proHB-EGF, in isolation from the diffusible form, can mediate cell signaling events. In addition, because effects of BAG-1 on regulated secretion of soluble HB-EGF were also identified, this interaction has the potential to alter the signaling capabilities of both the membrane-anchored and the diffusible forms of the growth factor.     

BAG-1蛋白及其对神经系统疾病调控研究进展

B细胞CLL/淋巴瘤2关联凋亡基因1（B-cell CLL/lymphoma 2-associated athanogene-1,BAG-1）编码的蛋白是一种多功能结合蛋白,包含不同功能的结构域,可与抗凋亡蛋白（Bcl-2）、热激蛋白70（heat shock protein 70,Hsp70/Hsc70）、细胞转化分裂介质（Raf-1）、类固醇激素受体和DNA等结合,对于调节细胞凋亡、信号传导、基因转录、细胞增殖与分化等具有重要作用。BAG-1参与多种神经系统疾病（如阿尔茨海默病、帕金森氏病、亨廷顿舞蹈病、脑中风、脊髓损伤以及神经精神障碍疾病）的发生发展过程。主要就BAG-1的结构、功能及其对神经系统疾病影响的研究进展进行了综述,以期为神经系统疾病的研究和治疗提供参考。     

Reversible inhibition of Hsp70 chaperone function by Scythe and Reaper

Protein folding mediated by the Hsp70 family of molecular chaperones requires both ATP and the co-chaperone Hdj-1. BAG-1 was recently identified as a bcl-2-interacting, anti-apoptotic protein that binds to the ATPase domain of Hsp70 and prevents the release of the substrate. While this suggested that cells had the potential to modulate Hsp70-mediated protein folding, physiological regulators of BAG-1 have yet to be identified. We report here that the apoptotic regulator Scythe, originally isolated through binding to the potent apoptotic inducer Reaper, shares limited sequence identity with BAG-1 and inhibits Hsp70- mediated protein refolding. Scythe-mediated inhibition of Hsp70 is reversed by Reaper, providing evidence for the regulated reversible inhibition of chaperone activity. As Scythe functions downstream of Reaper in apoptotic induction, these findings suggest that Scythe/Reaper may signal apoptosis, in part through regulating the folding and activity of apoptotic signaling molecules.     

Differential effects of the hsp70-binding protein BAG-1 on glucocorticoid receptor folding by the hsp90-based chaperone machinery

The heat shock protein hsp70/hsc70 is a required component of a five-protein (hsp90, hsp70, Hop, hsp40, and p23) minimal chaperone system reconstituted from reticulocyte lysate that forms glucocorticoid receptor (GR).hsp90 heterocomplexes. BAG-1 is a cofactor that binds to the ATPase domain of hsp70/hsc70 and that modulates its chaperone activity. Inasmuch as BAG-1 has been found in association with several members of the steroid receptor family, we have examined the effect of BAG-1 on GR folding and GR.hsp90 heterocomplex assembly. BAG-1 was present in reticulocyte lysate at a BAG-1:hsp70/hsc70 molar ratio of approximately 0.03, and its elimination by immunoadsorption did not affect GR folding and GR. hsp90 heterocomplex assembly. At low BAG-1:hsp70/hsc70 ratios, BAG-1 promoted the release of Hop from the hsp90-based chaperone system without inhibiting GR.hsp90 heterocomplex assembly. However, at molar ratios approaching stoichiometry with hsp70, BAG-1 produced a concentration-dependent inhibition of GR folding to the steroid-binding form with corresponding inhibition of GR.hsp90 heterocomplex assembly by the minimal five-protein chaperone system. Also, there was decreased steroid-binding activity in cells that were transiently or stably transfected with BAG-1. These observations suggest that, at physiological concentrations, BAG-1 modulates assembly by promoting Hop release from the assembly complex; but, at concentrations closer to those in transfected cells and some transformed cell lines, hsp70 is continuously bound by BAG-1, and heterocomplex assembly is blocked.     

BAG-1 modulates the chaperone activity of Hsp70/Hsc70.

The 70 kDa heat shock family of molecular chaperones is essential to a variety of cellular processes, yet it is unclear how these proteins are regulated in vivo. We present evidence that the protein BAG-1 is a potential modulator of the molecular chaperones, Hsp70 and Hsc70. BAG-1 binds to the ATPase domain of Hsp70 and Hsc70, without requirement for their carboxy-terminal peptide-binding domain, and can be co-immunoprecipitated with Hsp/Hsc70 from cell lysates. Purified BAG-1 and Hsp/Hsc70 efficiently form heteromeric complexes in vitro. BAG-1 inhibits Hsp/Hsc70-mediated in vitro refolding of an unfolded protein substrate, whereas BAG-1 mutants that fail to bind Hsp/Hsc70 do not affect chaperone activity. The binding of BAG-1 to one of its known cellular targets, Bcl-2, in cell lysates was found to be dependent on ATP, consistent with the possible involvement of Hsp/Hsc70 in complex formation. Overexpression of BAG-1 also protected certain cell lines from heat shock-induced cell death. The identification of Hsp/Hsc70 as a partner protein for BAG-1 may explain the diverse interactions observed between BAG-1 and several other proteins, including Raf-1, steroid hormone receptors and certain tyrosine kinase growth factor receptors. The inhibitory effects of BAG-1 on Hsp/Hsc70 chaperone activity suggest that BAG-1 represents a novel type of chaperone regulatory proteins and thus suggest a link between cell signaling, cell death and the stress response.     

The Carboxy-Terminal Domain of Hsc70 Provides Binding Sites for a Distinct Set of Chaperone Cofactors

The modulation of the chaperone activity of the heat shock cognate Hsc70 protein in mammalian cells involves cooperation with chaperone cofactors, such as Hsp40; BAG-1; the Hsc70-interacting protein, Hip; and the Hsc70-Hsp90-organizing protein, Hop. By employing the yeast two-hybrid system and in vitro interaction assays, we have provided insight into the structural basis that underlies Hsc70’s cooperation with different cofactors. The carboxy-terminal domain of Hsc70, previously shown to form a lid over the peptide binding pocket of the chaperone protein, mediates the interaction of Hsc70 with Hsp40 and Hop. Remarkably, the two cofactors bind to the carboxy terminus of Hsc70 in a noncompetitive manner, revealing the existence of distinct binding sites for Hsp40 and Hop within this domain. In contrast, Hip interacts exclusively with the amino-terminal ATPase domain of Hsc70. Hence, Hsc70 possesses separate nonoverlapping binding sites for Hsp40, Hip, and Hop. This appears to enable the chaperone protein to cooperate simultaneously with multiple cofactors. On the other hand, BAG-1 and Hip have recently been shown to compete in binding to the ATPase domain. Our data thus establish the existence of a network of cooperating and competing cofactors regulating the chaperone activity of Hsc70 in the mammalian cell.     

Short peptides derived from the BAG-1 C-terminus inhibit the interaction between BAG-1 and HSC70 and decrease breast cancer cell growth

BAG-1, a multifunctional protein, interacts with a plethora of cellular targets where the interaction with HSC70 and HSP70, is considered vital. Structural studies have demonstrated the C-terminal of BAG-1 forms a bundle of three alpha-helices of which helices 2 and 3 are directly involved in binding to the chaperones. Here we found peptides derived from helices 2 and 3 of BAG-1 interfered with BAG-1:HSC70 binding. We confirmed that a 12 amino-acid peptide from helix 2 directly interacted with HSC70 and when introduced into MCF-7 and ZR-75-1 cells, these peptides inhibited their growth. In conclusion, we have identified a small domain within BAG-1 which appears to play a critical role in the interaction with HSC70.

Structured summary

MINT-7265269, MINT-7265296, MINT-7265324, MINT-7265339, MINT-7265351, MINT-7265364, MINT-7265483, MINT-7265464, MINT-7265310: HSC70 (uniprotkb:P11142) binds (MI:0407) to BAG1 (uniprotkb:Q99933) by peptide array (MI:0081)MINT-7265281: peptide 15L (uniprotkb:Q99933) binds (MI:0407) to HSC70 (uniprotkb:P11142) by surface plasmon resonance (MI:0107)     

Down-regulation of Bcl-2-interacting protein BAG-1 confers resistance to anti-cancer drugs

BAG-1 was originally identified as a binding partner of anti-apoptotic factor Bcl-2 [Takayama et al., Cell 80 (1995) 279-284]. Exogenous expression of BAG-1 was reported to confer cells resistance to several stresses [Chen et al., Oncogene 21 (2002) 7050]. We have obtained human cervical cancer HeLa cells with down-regulated BAG-1 levels by using a highly specific and efficient RNA interference approach. Surprisingly, cells with down-regulated BAG-1 exhibited significantly lower sensitivity against several anti-cancer drugs than parental cells expressing normal levels of the protein. Furthermore, growth rate of the cells was reduced when BAG-1 was down-regulated. Activity of ERK pathway appeared to be decreased in BAG-1 down-regulated cells, as shown by the reduced phosphorylation of ERK1/2 proteins. Taken together resistance against anti-cancer drugs acquired by BAG-1 down-regulated cells may well be accounted for by the retardation of cell cycle progression, implicating the importance of BAG-1 in cell growth regulation.     

The ubiquitin-related BAG-1 provides a link between the molecular chaperones Hsc70/Hsp70 and the proteasome

The BAG-1 protein modulates the chaperone activity of Hsc70 and Hsp70 in the mammalian cytosol and nucleus. Remarkably, BAG-1 possesses a ubiquitin-like domain at its amino terminus, suggesting a link to the ubiquitin/proteasome system. Here we show that BAG-1 is indeed associated with the 26 S proteasome in HeLa cells. Binding of the chaperone cofactor to the proteolytic complex is regulated by ATP hydrolysis and is not mediated by Hsc70 and Hsp70. The presented findings reveal a role of BAG-1 as a physical link between the Hsc70/Hsp70 chaperone system and the proteasome. In fact, targeting of BAG-1 to the proteasome promotes an association of the chaperones with the proteolytic complex in vitro and in vivo. A regulatory function of the chaperone cofactor at the interface between protein folding and protein degradation is thus indicated.