Molecular Chaperone Hsc70 Research Articles

C-Myc, AKT, Hsc70, and the T-Box Transcription Factor TBX3 Form an Important Oncogenic Signaling Axis in Breast Cancer.

Breast cancer is the second leading cause of death in women globally, and it remains a health burden due to poor therapy response, cancer cell drug resistance, and the debilitating side effects associated with most therapies. One approach to addressing the need to improve breast cancer therapies has been to elucidate the mechanism(s) underpinning this disease to identify key drivers that can be targeted in molecular therapies. The T-box transcription factor, TBX3, is upregulated in breast cancer, in which it contributes to important oncogenic processes, and it has been validated as a potential therapeutic target. Here, we investigated the molecular mechanisms that upregulate TBX3 in breast cancer, and we show that it involves transcriptional activation by c-Myc, post-translational modification by AKT1 and AKT3, and interaction with the molecular chaperone Hsc70. Together, the results from this study provide evidence that c-Myc, AKT, Hsc70, and TBX3 form part of an important oncogenic pathway in breast cancer and thus reveal versatile ways of interfering with the oncogenic activity of TBX3 for the treatment of this neoplasm. Implications: Targeting the c-Myc/AKT/TBX3/Hsc70 signaling axis may be an effective treatment strategy for TBX3-driven breast cancer.

Quercetin and HSC70 coregulate the anti-inflammatory action of the ubiquitin-like protein MNSFβ.

Quercetin is a flavonol that modifies many cellular processes. Monoclonal nonspecific suppressor factor β is a member of the ubiquitin-like family of proteins that are involved in various biological processes. It has been demonstrated that quercetin regulates the effect of MNSFβ on tumor necrosis factor-α secretion in lipopolysaccharide (LPS)-stimulated macrophages. This study found that quercetin and the heat shock protein HSC70 coregulate the action of MNSFβ. Quercetin dose-dependently suppressed the LPS/interferon γ-induced nitric oxide production without cytotoxicity in the macrophage-like cell line Raw264.7. SiRNA knockdown experiments showed that quercetin inhibited the MNSFβ and HSC70 siRNA-mediated enhancement of TNFα and the production of RANTES, a member of C-C chemokine superfamily, in LPS-stimulated Raw264.7 cells. Western blot analysis showed that quercetin and HSC70 regulated ERK1/2 activation and LPS-stimulated IκBα degradation by affecting the complex formation of MNSFβ and the proapoptotic protein Bcl-G. Moreover, MNSFβ is implicated in TLR4/MyD88 signaling but not in TLR3 signaling. HSC70 is an important chaperone that facilitates the stabilization of MNSFβ. Quercetin may negatively control the function of MNSFβ by regulating the action of the molecular chaperone HSC70. MNSFβ mediates TLR4/Myd88 signaling but not TLR3 signaling.

Nematode CDC-37 and DNJ-13 form complexes and can interact with HSP-90

The molecular chaperones Hsc70 and Hsp90 are required for proteostasis control and specific folding of client proteins in eukaryotic and prokaryotic organisms. Especially in eukaryotes these ATP-driven molecular chaperones are interacting with cofactors that specify the client spectrum and coordinate the ATPase cycles. Here we find that a Hsc70-cofactor of the Hsp40 family from nematodes, DNJ-13, directly interacts with the kinase-specific Hsp90-cofactor CDC-37. The interaction is specific for DNJ-13, while DNJ-12 another DnaJ-like protein of C. elegans, does not bind to CDC-37 in a similar manner. Analytical ultracentrifugation is employed to show that one CDC-37 molecule binds to a dimeric DNJ-13 protein with low micromolar affinity. We perform cross-linking studies with mass spectrometry to identify the interaction site and obtain specific cross-links connecting the N-terminal J-domain of DNJ-13 with the N-terminal domain of CDC-37. Further AUC experiments reveal that both, the N-terminal part of CDC-37 and the C-terminal domain of CDC-37, are required for efficient interaction. Furthermore, the presence of DNJ-13 strengthens the complex formation between CDC-37 and HSP-90 and modulates the nucleotide-dependent effects. These findings on the interaction between Hsp40 proteins and Hsp90-cofactors provide evidence for a more intricate interaction between the two chaperone systems during client processing.

Polypeptides derived from α-Synuclein binding partners to prevent α-Synuclein fibrils interaction with and take-up by cells.

α-Synuclein (αSyn) fibrils spread from one neuronal cell to another. This prion-like phenomenon is believed to contribute to the progression of the pathology in Parkinson's disease and other synucleinopathies. The binding of αSyn fibrils originating from affected cells to the plasma membrane of naïve cells is key in their prion-like propagation propensity. To interfere with this process, we designed polypeptides derived from proteins we previously showed to interact with αSyn fibrils, namely the molecular chaperone Hsc70 and the sodium/potassium pump NaK-ATPase and assessed their capacity to bind αSyn fibrils and/or interfere with their take-up by cells of neuronal origin. We demonstrate here that polypeptides that coat αSyn fibrils surfaces in such a way that they are changed affect αSyn fibrils binding to the plasma membrane components and/or their take-up by cells. Altogether our observations suggest that the rationale design of αSyn fibrils polypeptide binders that interfere with their propagation between neuronal cells holds therapeutic potential.

Interaction of the chaperones alpha B-crystallin and CHIP with fibrillar alpha-synuclein: Effects on internalization by cells and identification of interacting interfaces

The spread of fibrillar alpha-synuclein from affected to naïve neuronal cells is thought to contribute to the progression of synucleinopathies. The binding of fibrillar alpha-synuclein to the plasma membrane is key in this process. We and others previously showed that coating fibrillar alpha-synuclein by the molecular chaperone Hsc70 affects fibrils properties. Here we assessed the effect of the two molecular chaperones alpha B-crystallin and CHIP on alpha-synuclein fibrils uptake by Neuro-2a cells. We demonstrate that both chaperones diminish fibrils take up by cells. We identify through a cross-linking and mass spectrometry strategy the interaction interfaces between alpha-synuclein fibrils and alpha B-crystallin or CHIP. Our results open the way for designing chaperone-derived polypeptide binders that interfere with the propagation of pathogenic alpha-synuclein assemblies.

Head-bent resistant Hsc70 variants show reduced Hsp40 affinity and altered protein folding activity

The molecular chaperone Hsc70 performs essential tasks by folding proteins. Hsc70 is driven by the hydrolysis of ATP and tuned by the association with various co-chaperones. One such cofactor is the nematode nucleotide exchange factor UNC-23, whose mutation disrupts muscle attachment and induces a severe head-bent phenotype in C.elegans. Interestingly, four mutations in Hsc70 can suppress this phenotype, but the molecular mechanism underlying this suppression is unknown. Here we characterize these four suppressor variants, Hsc70 D233N, S321F, A379V and D384N. In vitro only Hsc70 S321F shows reduced stability and altered nucleotide interaction, but all mutations affect the ATPase stimulation. In particular, Hsc70 D233N and Hsc70 A379V show strongly reduced interactions with DNJ-12 and DNJ-13. Nucleotide exchange factor binding instead is barely influenced in Hsc70 D233N, A379V and D384N and their chaperone activity is preserved. Molecular dynamics simulations suggest that effects in Hsc70 S321F and Hsc70 A379V originate from steric clashes in the vicinity of the mutation site, while D233N disrupts a salt bridge that contributes to Hsc70’s nucleotide-induced conformational changes. In summary, the analyzed mutants show altered ATPase and refolding activity caused by changes in Hsp40 binding.

Abstract 385: Cardiac Myosin Binding Protein-C Mutants Disrupt Ubiquitin Proteasome System and Hsc70 Functions

The most commonly mutated gene in hypertrophic cardiomyopathy (HCM) is cardiac myosin binding protein C (MYBPC3). Over 90% of MYBPC3 mutations are nonsense, but whether these mutations manifest in loss- or gain-of-function is unresolved. Evidence suggests MYBPC3 mutants impact protein quality control mechanisms. The objective of this study was to evaluate interactions of MYBPC3 with proteostatic systems and test the hypothesis that these interactions affect protein homeostasis in cardiomyocytes. WT and mutant MYBPC3 constructs were expressed in neonatal rat ventricular myocytes (NRVMs) via adenovirus. Mutant MYBPC3 induced ubiquitin proteasome system reporter GFPu accumulation (fold increase in GFPu-positive cells vs control: WT 138±14.0%, mutant 198±27.2%, mean±SEM, p<0.05 vs control and WT), indicating proteasome dysfunction. Affinity purification/mass spectrometry identified molecular chaperones Hsp70 and Hsc70 as prominent interactors with MYBPC3. We observed MYBPC3 degradation by cycloheximide chase in response to Hsc70 siRNA knockdown or pharmacological treatment with Hsp70 activator YM-1. Hsc70 knockdown slowed degradation of WT and mutant MYBPC3 (WT control t ½ =5.47±0.70 hr, WT Hsc70 knockdown t ½ =13.5±1.62 hr; mutant control t ½ =3.42±0.61 hr, mutant Hsc70 knockdown t ½ =9.87±0.95 hr), while YM-1 treatment accelerated degradation (WT DMSO t ½ =10.2±3.28 hr, WT YM-1 t ½ =3.16±0.61 hr; mutant DMSO t ½ =11.7±2.67 hr, mutant YM-1 t ½ =1.37±0.16 hr). We then evaluated whether transferrin uptake via clathrin mediated endocytosis, a critical Hsc70-dependent activity, was affected by mutant MYBPC3. Transferrin uptake was significantly decreased in NRVMs expressing mutant MYBPC3 compared to WT and untreated controls (transferrin-positive cells: control 22.93±3.34%, WT 17.47±0.70%, mutant 9.30±1.63%, mean±SEM, p<0.05 vs control and WT). In conclusion, we have demonstrated that Hsp70 chaperones interact with MYBPC3 in cardiomyocytes and affect MYBPC3 degradation, suggesting MYBPC3 is a client of Hsp70 and Hsc70. Additionally, expression of mutant MYBPC3 causes ubiquitin proteasome impairment and interferes with normal Hsc70 function. These results support our hypothesis that mutant MYBPC3 affects protein homeostasis in HCM.

Molecular Chaperone Hsp70 and Its Constitutively Active Form Hsc70 Play an Indispensable Role During Eye Development of Drosophila melanogaster.

In the present study, we demonstrate that molecular chaperone Hsp70 and Hsc70 is essential for normal organization and development of ommatidial cells in Drosophila melanogaster eye. An exogenously expressed dominant negative mutant of Hsp70 (K71E) and Hsc70.4 (K71S and D206S) in an eye-specific manner resulted in eye degeneration that includes loss of eye pigment, disorganized ommatidia, abnormality in bristle cell arrangement and reduction in the eye size. The developmental organization of ommatidial cells (cone, photoreceptor, pigment, and bristle cell complex) was disturbed in Hsp70 and Hsc70 mutants. Acridine orange (AO) and caspase 3 staining showed an increased cell death in Hsp70 and Hsc70 mutant eyes. Genetic interaction study of Hsp70 and Hsc70 mutants with candidate genes of JNK signaling pathway and immunocytochemistry study using phospho-JNK antibody suggested that mutation in Hsp70 and Hsc70 results in ectopic activation of JNK signaling in fly eye. Further, anti-PH3 staining in Hsp70 and Hsc70 mutant eyes revealed a reduced number of mitotic cells in second mitotic wave (SMW) of developing eye and anti-Rh1 staining showed reduced Rh1 expression, accumulation of Rh1 in the cytoplasm, and rhabdomere degeneration. Thus, on the basis of results, it was concluded that molecular chaperone Hsp70 and Hsc70 play an indispensable role during Drosophila eye development.

Regulation of CLC-1 chloride channel biosynthesis by FKBP8 and Hsp90β.

Mutations in human CLC-1 chloride channel are associated with the skeletal muscle disorder myotonia congenita. The disease-causing mutant A531V manifests enhanced proteasomal degradation of CLC-1. We recently found that CLC-1 degradation is mediated by cullin 4 ubiquitin ligase complex. It is currently unclear how quality control and protein degradation systems coordinate with each other to process the biosynthesis of CLC-1. Herein we aim to ascertain the molecular nature of the protein quality control system for CLC-1. We identified three CLC-1-interacting proteins that are well-known heat shock protein 90 (Hsp90)-associated co-chaperones: FK506-binding protein 8 (FKBP8), activator of Hsp90 ATPase homolog 1 (Aha1), and Hsp70/Hsp90 organizing protein (HOP). These co-chaperones promote both the protein level and the functional expression of CLC-1 wild-type and A531V mutant. CLC-1 biosynthesis is also facilitated by the molecular chaperones Hsc70 and Hsp90β. The protein stability of CLC-1 is notably increased by FKBP8 and the Hsp90β inhibitor 17-allylamino-17-demethoxygeldanamycin (17-AAG) that substantially suppresses cullin 4 expression. We further confirmed that cullin 4 may interact with Hsp90β and FKBP8. Our data are consistent with the idea that FKBP8 and Hsp90β play an essential role in the late phase of CLC-1 quality control by dynamically coordinating protein folding and degradation.

The Molecular Chaperone Hsc70 Interacts with Tyrosine Hydroxylase to Regulate Enzyme Activity and Synaptic Vesicle Localization

We previously reported that the vesicular monoamine transporter 2 (VMAT2) is physically and functionally coupled with Hsc70 as well as with the dopamine synthesis enzymes tyrosine hydroxylase (TH) and aromatic amino acid decarboxylase, providing a novel mechanism for dopamine homeostasis regulation. Here we expand those findings to demonstrate that Hsc70 physically and functionally interacts with TH to regulate the enzyme activity and synaptic vesicle targeting. Co-immunoprecipitation assays performed in brain tissue and heterologous cells demonstrated that Hsc70 interacts with TH and aromatic amino acid decarboxylase. Furthermore, in vitro binding assays showed that TH directly binds the substrate binding and carboxyl-terminal domains of Hsc70. Immunocytochemical studies indicated that Hsc70 and TH co-localize in midbrain dopaminergic neurons. The functional significance of the Hsc70-TH interaction was then investigated using TH activity assays. In both dopaminergic MN9D cells and mouse brain synaptic vesicles, purified Hsc70 facilitated an increase in TH activity. Neither the closely related protein Hsp70 nor the unrelated Hsp60 altered TH activity, confirming the specificity of the Hsc70 effect. Overexpression of Hsc70 in dopaminergic MN9D cells consistently resulted in increased TH activity whereas knockdown of Hsc70 by short hairpin RNA resulted in decreased TH activity and dopamine levels. Finally, in cells with reduced levels of Hsc70, the amount of TH associated with synaptic vesicles was decreased. This effect was rescued by addition of purified Hsc70. Together, these data demonstrate a novel interaction between Hsc70 and TH that regulates the activity and localization of the enzyme to synaptic vesicles, suggesting an important role for Hsc70 in dopamine homeostasis.

DnaJ/Hsc70 chaperone complexes control the extracellular release of neurodegenerative-associated proteins.

It is now known that proteins associated with neurodegenerative disease can spread throughout the brain in a prionlike manner. However, the mechanisms regulating the trans-synaptic spread propagation, including the neuronal release of these proteins, remain unknown. The interaction of neurodegenerative disease-associated proteins with the molecular chaperone Hsc70 is well known, and we hypothesized that much like disaggregation, refolding, degradation, and even normal function, Hsc70 may dictate the extracellular fate of these proteins. Here, we show that several proteins, including TDP-43, α-synuclein, and the microtubule-associated protein tau, can be driven out of the cell by an Hsc70 co-chaperone, DnaJC5. In fact, DnaJC5 overexpression induced tau release in cells, neurons, and brain tissue, but only when activity of the chaperone Hsc70 was intact and when tau was able to associate with this chaperone. Moreover, release of tau from neurons was reduced in mice lacking the DnaJC5 gene and when the complement of DnaJs in the cell was altered. These results demonstrate that the dynamics of DnaJ/Hsc70 complexes are critically involved in the release of neurodegenerative disease proteins.

Methionine to isothreonine conversion as a source of false discovery identifications of genetically encoded variants in proteogenomics.

A lot of effort is currently put into proteogenomics of cancer. Studies detect non-synonymous cancer mutations at protein level by search of high-throughput LC-MS/MS data against customized genomic databases. In such studies, much attention is paid to potential false positive identifications. Here we describe one possible cause of such false identifications, an artifact of sample preparation which mimics methionine to threonine nucleic acid-encoded variant. The methionine to isothreonine conversion should be taken into consideration for correct interpretation of proteogenomic data.

A novel bio-orthogonal cross-linker for improved protein/protein interaction analysis.

The variety of protein cross-linkers developed in recent years illustrates the current requirement for efficient reagents optimized for mass spectrometry (MS) analysis. To date, the most widely used strategy relies on commercial cross-linkers that bear an isotopically labeled tag and N-hydroxysuccinimid-ester (NHS-ester) moieties. Moreover, an enrichment step using liquid chromatography is usually performed after enzymatic digestion of the cross-linked proteins. Unfortunately, this approach suffers from several limitations. First, it requires large amounts of proteins. Second, NHS-ester cross-linkers are poorly efficient because of their fast hydrolysis in water. Finally, data analysis is complicated because of uneven fragmentation of complex isotopic cross-linked peptide mixtures. We therefore synthesized a new type of trifunctional cross-linker to overrule these limitations. This reagent, named NNP9, comprises a rigid core and bears two activated carbamate moieties and an azido group. NNP9 was used to establish intra- and intermolecular cross-links within creatine kinase, then to map the interaction surfaces between α-Synuclein (α-Syn), the aggregation of which leads to Parkinson's disease, and the molecular chaperone Hsc70. We show that NNP9 cross-linking efficiency is significantly higher than that of NHS-ester commercial cross-linkers. The number of cross-linked peptides identified was increased, and a high quality of MS/MS spectra leading to high sequence coverage was observed. Our data demonstrate the potential of NNP9 for an efficient and straightforward characterization of protein-protein interfaces and illustrate the power of using different cross-linkers to map thoroughly the surface interfaces within protein complexes.

Molecular Interaction between the Chaperone Hsc70 and the N-terminal Flank of Huntingtin Exon 1 Modulates Aggregation

The aggregation of polyglutamine (polyQ)-containing proteins is at the origin of nine neurodegenerative diseases. Molecular chaperones prevent the aggregation of polyQ-containing proteins. The exact mechanism by which they interact with polyQ-containing, aggregation-prone proteins and interfere with their assembly is unknown. Here we dissect the mechanism of interaction between a huntingtin exon 1 fragment of increasing polyQ lengths (HttEx1Qn), the aggregation of which is tightly associated with Huntington's disease, and molecular chaperone Hsc70. We show that Hsc70, together with its Hsp40 co-chaperones, inhibits HttEx1Qn aggregation and modifies the structural, seeding, and infectious properties of the resulting fibrils in a polyQ-independent manner. We demonstrate that Hsc70 binds the 17-residue-long N-terminal flank of HttEx1Qn, and we map Hsc70-HttEx1Qn surface interfaces at the residue level. Finally, we show that this interaction competes with homotypic interactions between the N termini of different HttEx1Qn molecules that trigger the aggregation process. Our results lay the foundations of future therapeutic strategies targeting huntingtin aggregation in Huntington disease.

The Balanced Regulation of Hsc70 by DNJ-13 and UNC-23 Is Required for Muscle Functionality

The molecular chaperone Hsc70 assists in the folding of non-native proteins together with its J domain- and BAG domain-containing cofactors. In Caenorhabditis elegans, two BAG domain-containing proteins can be identified, one of them being UNC-23, whose mutation induces severe motility dysfunctions. Using reporter strains, we find that the full-length UNC-23, in contrast to C-terminal fragments, localizes specifically to the muscular attachment sites. C-terminal fragments of UNC-23 instead perform all Hsc70-related functions, like ATPase stimulation and regulation of folding activity, albeit with lower affinity than BAG-1. Interestingly, overexpression of CFP-Hsc70 can induce muscular defects in wild-type nematodes that phenocopy the knockout of its cofactor UNC-23. Strikingly, the motility dysfunction in the unc-23 mutated strain can be cured specifically by down-regulation of the antagonistic Hsc70 cochaperone DNJ-13, implying that the severe phenotype is caused by misregulation of the Hsc70 cycle. These findings point out that the balanced action of cofactors in the ATP-driven cycle of Hsc70 is crucial for the contribution of Hsc70 to muscle functionality.

The E3 Ubiquitin Ligase CHIP and the Molecular Chaperone Hsc70 Form a Dynamic, Tethered Complex

The E3 ubiquitin ligase CHIP (C-terminus of Hsc70 Interacting Protein, a 70 kDa homodimer) binds to the molecular chaperone Hsc70 (a 70 kDa monomer), and this complex is important in both the ubiquitination of Hsc70 and the turnover of Hsc70-bound clients. Here we used NMR spectroscopy, biolayer interferometry, and fluorescence polarization to characterize the Hsc70-CHIP interaction. We found that CHIP binds tightly to two molecules of Hsc70 forming a 210 kDa complex, with a Kd of approximately 60 nM, and that the IEEVD motif at the C-terminus of Hsc70 (residues 642-646) is both necessary and sufficient for binding. Moreover, the same motif is required for CHIP-mediated ubiquitination of Hsc70 in vitro, highlighting its functional importance. Relaxation-based NMR experiments on the Hsc70-CHIP complex determined that the two partners move independently in solution, similar to "beads on a string". These results suggest that a dynamic C-terminal region of Hsc70 provides for flexibility between CHIP and the chaperone, allowing the ligase to "search" a large space and engage in productive interactions with a wide range of clients. In support of this suggestion, we find that deleting residues 623-641 of the C-terminal region, while retaining the IEEVD motif, caused a significant decrease in the efficiency of Hsc70 ubiquitination by CHIP.

Molecular chaperone Hsp110 rescues a vesicle transport defect produced by an ALS-associated mutant SOD1 protein in squid axoplasm

Mutant human Cu/Zn superoxide dismutase 1 (SOD1) is associated with motor neuron toxicity and death in an inherited form of amyotrophic lateral sclerosis (ALS; Lou Gehrig disease). One aspect of toxicity in motor neurons involves diminished fast axonal transport, observed both in transgenic mice and, more recently, in axoplasm isolated from squid giant axons. The latter effect appears to be directly mediated by misfolded SOD1, whose addition activates phosphorylation of p38 MAPK and phosphorylation of kinesin. Here, we observe that several different oligomeric states of a fusion protein, comprising ALS-associated human G85R SOD1 joined with yellow fluorescent protein (G85R SOD1YFP), which produces ALS in transgenic mice, inhibited anterograde transport when added to squid axoplasm. Inhibition was blocked both by an apoptosis signal-regulating kinase 1 (ASK1; MAPKKK) inhibitor and by a p38 inhibitor, indicating the transport defect is mediated through the MAPK cascade. In further incubations, we observed that addition of the mammalian molecular chaperone Hsc70, abundantly associated with G85R SOD1YFP in spinal cord of transgenic mice, exerted partial correction of the transport defect, associated with diminished phosphorylation of p38. Most striking, the addition of the molecular chaperone Hsp110, in a concentration substoichiometric to the mutant SOD1 protein, completely rescued both the transport defect and the phosphorylation of p38. Hsp110 has been demonstrated to act as a nucleotide exchange factor for Hsc70 and, more recently, to be able to cooperate with it to mediate protein disaggregation. We speculate that it can cooperate with endogenous squid Hsp(c)70 to mediate binding and/or disaggregation of mutant SOD1 protein, abrogating toxicity.

Cellular Mechanotransduction Relies on Tension-Induced and Chaperone-Assisted Autophagy

Mechanical tension is an ever-present physiological stimulus essential for the development and homeostasis of locomotory, cardiovascular, respiratory, and urogenital systems. Tension sensing contributes to stem cell differentiation, immune cell recruitment, and tumorigenesis. Yet, how mechanical signals are transduced inside cells remains poorly understood. Here, we identify chaperone-assisted selective autophagy (CASA) as a tension-induced autophagy pathway essential for mechanotransduction in muscle and immune cells. The CASA complex, comprised of the molecular chaperones Hsc70 and HspB8 and the cochaperone BAG3, senses the mechanical unfolding of the actin-crosslinking protein filamin. Together with the chaperone-associated ubiquitin ligase CHIP, the complex initiates the ubiquitin-dependent autophagic sorting of damaged filamin to lysosomes for degradation. Autophagosome formation during CASA depends on an interaction of BAG3 with synaptopodin-2 (SYNPO2). This interaction is mediated by the BAG3 WW domain and facilitates cooperation with an autophagosome membrane fusion complex. BAG3 also utilizes its WW domain to engage in YAP/TAZ signaling. Via this pathway, BAG3 stimulates filamin transcription to maintain actin anchoring and crosslinking under mechanical tension. By integrating tension sensing, autophagosome formation, and transcription regulation during mechanotransduction, the CASA machinery ensures tissue homeostasis and regulates fundamental cellular processes such as adhesion, migration, and proliferation.

Matrine modulates HSC70 levels and rescues ΔF508‐CFTR

Cystic fibrosis transmembrane conductance regulator (CFTR) is a cAMP-dependent Cl(-) channel located in the plasma membrane, and its malfunction results in cystic fibrosis (CF), the most common lethal genetic disease in Caucasians. Most CF patients carry the deletion of Phe508 (ΔF508 mutation); this mutation prevents the delivery of the CFTR to its correct cellular location, the apical (lumen-facing) membrane of epithelial cells. Molecular chaperones play a central role in determining the fate of ΔF508-CFTR. In this report, we show that the Matrine, a quinolizidine alkaloid, downregulates the expression of the molecular chaperone HSC70 and increases the protein levels of ΔF508-CFTR in human alveolar basal epithelial cells (A549 cell line), stably transfected with a ΔF508-CFTR-expressing construct. Moreover, Matrine induced ΔF508-CFTR release from endoplasmic reticulum to cell cytosol and its localization on the cell membrane. Interestingly, downregulation of HSC70 resulted in increased levels of ΔF508-CFTR complexes with the co-chaperone BAG3 that in addition appeared to co-localize with the mutated protein on the cell surface. These results shed new light on ΔF508-CFTR interactions with proteins of the chaperones/co-chaperones system and could be useful in strategies for future medical treatments for CF.

Methylglyoxal Alters the Function and Stability of Critical Components of the Protein Quality Control

BackgroundIncreased production and accumulation of methylglyoxal (MGO), as well as increased modification of proteins by glycoxidation, are hallmarks of aging and diabetes. MGO was shown to modify proteins and to contribute to the accumulation of damaged proteins that can be toxic to cells. However, the effect of MGO on the cell systems responsible for repairing or degrading damaged proteins is still unclear. In this study, the effect of MGO on the function of the ubiquitin-proteasome system (UPS) and on molecular chaperones, two cooperative mechanisms associated with protein quality control, was investigated.Principal FindingsIn this work it is shown that treatment of cells with MGO leads to accumulation of ubiquitin conjugates and depletion of free ubiquitin. Moreover, MGO significantly decreases the proteolytic activity of the 20S proteasome. Data further shows that MGO decreases the levels of the molecular chaperones Hsc70 and Hsp90 and leads to accumulation of CHIP-, Hsp40- and ubiquitin-containing aggregates. The formation of large aggregates containing CHIP is a consequence of its binding to misfolded proteins and to molecular chaperones. Moreover, dysfunction of the chaperones/CHIP/UPS axis is associated with accumulation of oxidized and argpyrimidine-modified proteins, which is likely to be associated with decreased cell viability. Interestingly, data further shows that MGO-induced stress induces the activation of heat shock factor-1 (Hsf-1), the main transcription factor involved in the regulation of the expression of heat shock proteins (HSPs) and cell response to stress.ConclusionsThe data obtained in this work suggests that MGO impairs both the UPS and the protein quality control dependent on CHIP and molecular chaperones, leading to accumulation of toxic aggregates and increased cell death. However, these MGO-induced changes appear to elicit a response from the Hsf-1 system, which is crucial to help cells to cope with cellular stress and to re-establish homeostasis.