Most currently available small molecule inhibitors of DNA replication lack enzymatic specificity, resulting in deleterious side effects during use in cancer chemotherapy and limited experimental usefulness as mechanistic tools to study DNA replication. Towards development of targeted replication inhibitors, we have focused on Mcm2-7 (minichromosome maintenance protein 2–7), a highly conserved helicase and key regulatory component of eukaryotic DNA replication. Unexpectedly we found that the fluoroquinolone antibiotic ciprofloxacin preferentially inhibits Mcm2-7. Ciprofloxacin blocks the DNA helicase activity of Mcm2-7 at concentrations that have little effect on other tested helicases and prevents the proliferation of both yeast and human cells at concentrations similar to those that inhibit DNA unwinding. Moreover, a previously characterized mcm mutant (mcm4chaos3) exhibits increased ciprofloxacin resistance. To identify more potent Mcm2-7 inhibitors, we screened molecules that are structurally related to ciprofloxacin and identified several that compromise the Mcm2-7 helicase activity at lower concentrations. Our results indicate that ciprofloxacin targets Mcm2-7 in vitro, and support the feasibility of developing specific quinolone-based inhibitors of Mcm2-7 for therapeutic and experimental applications.
AAA+ ATPase; DNA replication; fluoroquinolone; helicase; minichromosome maintenance complex; CI, confidence interval; DCCD, N,N′-dicyclohexylcarbodi-imide; EEDQ, N-ethoxy-carbonyl-2-ethoxy-1,2-dyhydroquinolone; Mcm, minichromosome maintenance protein; Nbf, 4-chloro-7-nitrobenzofurazan; NEM, N-ethylmaleimide; PG, phenylglyoxal; PP, pyridoxal phosphate; SsoMcm, Sulfolobus solfataricus Mcm protein; SV40, simian virus 40; TAg, T-antigen; Topo I, topoisomerase I; Topo II, topoisomerase II
New polyomaviruses are continually being identified, and it is likely that links between this virus family and disease will continue to emerge. Unfortunately, a specific treatment for polyomavirus-associated disease is lacking. Because polyomaviruses express large Tumor Antigen, TAg, we hypothesized that small molecule inhibitors of the essential ATPase activity of TAg would inhibit viral replication. Using a new screening platform, we identified inhibitors of TAg's ATPase activity. Lead compounds were moved into a secondary assay, and ultimately two FDA approved compounds, bithionol and hexachlorophene, were identified as the most potent TAg inhibitors known to date. Both compounds inhibited Simian Virus 40 replication as assessed by plaque assay and quantitative PCR. Moreover, these compounds inhibited BK virus, which causes BKV Associated Nephropathy. In neither case was host cell viability compromised at these concentrations. Our data indicate that directed screening for TAg inhibitors is a viable method to identify polyomavirus inhibitors, and that bithionol and hexachlorophene represent lead compounds that may be further modified and/or ultimately used to combat diseases associated with polyomavirus infection.
polyomavirus; bithionol; hexachlorophene; T antigen; molecular chaperone; high throughput screen
Ubiquitylation of partially misfolded proteins by the yeast Doa10 E3 ligase requires the Hsp40 cochaperone Sis1, whereas the Hsp70 chaperones Ssa1 and Ssa2 are dispensable. Elimination of the Hsp70 chaperones prevents proteasomal degradation, resulting in ubiquitin-dependent sequestration of the misfolded proteins in Hsp42-positive foci.
Ubiquitin accumulation in amyloid plaques is a pathological marker observed in the vast majority of neurodegenerative diseases, yet ubiquitin function in these inclusions is controversial. It has been suggested that ubiquitylated proteins are directed to inclusion bodies under stress conditions, when both chaperone-mediated refolding and proteasomal degradation are compromised or overwhelmed. Alternatively, ubiquitin and chaperones may be recruited to preformed inclusions to promote their elimination. We address this issue using a yeast model system, based on expression of several mildly misfolded degradation substrates in cells with altered chaperone content. We find that the heat shock protein 70 (Hsp70) chaperone pair Ssa1/Ssa2 and the Hsp40 cochaperone Sis1 are essential for degradation. Substrate ubiquitylation is strictly dependent on Sis1, whereas Ssa1 and Ssa2 are dispensable. Remarkably, in Ssa1/Ssa2-depleted cells, ubiquitylated substrates are sequestered into detergent-insoluble, Hsp42-positive inclusion bodies. Unexpectedly, sequestration is abolished by preventing substrate ubiquitylation. We conclude that Hsp40 is required for the targeting of misfolded proteins to the ubiquitylation machinery, whereas the decision to degrade or sequester ubiquitylated proteins is mediated by the Hsp70s. Accordingly, diminished Hsp70 levels, as observed in aging or certain pathological conditions, might be sufficient to trigger ubiquitin-dependent sequestration of partially misfolded proteins into inclusion bodies.
Inactivation of Cdc48p/p97 triggers formation of a complex that contains the 26S proteasome, Cdc48p/p97, ubiquitinated substrates, select components of the Hrd1 complex, and the lumenal recognition factor, Yos9p. A model is proposed in which the Hrd1 complex links substrate recognition and degradation on opposite sides of the ER membrane.
During endoplasmic reticulum–associated degradation (ERAD), misfolded lumenal and membrane proteins in the ER are recognized by the transmembrane Hrd1 ubiquitin ligase complex and retrotranslocated to the cytosol for ubiquitination and degradation. Although substrates are believed to be delivered to the proteasome only after the ATPase Cdc48p/p97 acts, there is limited knowledge about how the Hrd1 complex coordinates with Cdc48p/p97 and the proteasome to orchestrate substrate recognition and degradation. Here we provide evidence that inactivation of Cdc48p/p97 stalls retrotranslocation and triggers formation of a complex that contains the 26S proteasome, Cdc48p/p97, ubiquitinated substrates, select components of the Hrd1 complex, and the lumenal recognition factor, Yos9p. We propose that the actions of Cdc48p/p97 and the proteasome are tightly coupled during ERAD. Our data also support a model in which the Hrd1 complex links substrate recognition and degradation on opposite sides of the ER membrane.
The authors conducted a high-throughput screening campaign for inhibitors of SV40 large T antigen ATPase activity to identify candidate antivirals that target the replication of polyomaviruses. The primary assay was adapted to 1536-well microplates and used to screen the National Institutes of Health Molecular Libraries Probe Centers Network library of 306 015 compounds. The primary screen had an Z value of ~0.68, signal/background = 3, and a high (5%) DMSO tolerance. Two counterscreens and two secondary assays were used to prioritize hits by EC50, cytotoxicity, target specificity, and off-target effects. Hits that inhibited ATPase activity by >44% in the primary screen were tested in dose–response efficacy and eukaryotic cytotoxicity assays. After evaluation of hit cytotoxicity, drug likeness, promiscuity, and target specificity, three compounds were chosen for chemical optimization. Chemical optimization identified a class of bisphenols as the most effective biochemical inhibitors. Bisphenol A inhibited SV40 large T antigen ATPase activity with an IC50 of 41 μM in the primary assay and 6.2 μM in a cytoprotection assay. This compound class is suitable as probes for biochemical investigation of large T antigen ATPase activity, but because of their cytotoxicity, further optimization is necessary for their use in studying polyomavirus replication in vivo.
SV40; chemistry; large T antigen; HTS; antivirals
Merkel cell polyomavirus (MCV) causes ~80% of primary and metastatic Merkel cell carcinomas (MCCs). By comparing digital transcriptome subtraction deep-sequencing profiles, we found that transcripts of the cellular survivin oncoprotein [BIRC5a (baculoviral inhibitor of apoptosis repeat-containing 5)] were up-regulated sevenfold in virus-positive compared to virus-negative MCC tumors. Knockdown of MCV large T antigen in MCV-positive MCC cell lines decreased survivin mRNA and protein expression. Exogenously expressed MCV large T antigen increased survivin protein expression in non-MCC primary cells. This required an intact retinoblastoma protein–targeting domain that activated survivin gene transcription as well as expression of other G1-S–phase proteins including E2F1 and cyclin E. Survivin expression is critical to the survival of MCV-positive MCC cells. A small-molecule survivin inhibitor, YM155, potently and selectively initiates irreversible, nonapoptotic, programmed MCV-positive MCC cell death. Of 1360 other chemotherapeutic and pharmacologically active compounds screened in vitro, only bortezomib (Velcade) was found to be similarly potent, but was not selective in killing MCV-positive MCC cells. YM155 halted the growth of MCV-positive MCC xenograft tumors and was nontoxic in mice, whereas bortezomib was not active in vivo and mice displayed serious morbidity. Xenograft tumors resumed growth once YM155 treatment was stopped, suggesting that YM155 may be cytostatic rather than cytotoxic in vivo. Identifying the cellular pathways, such as those involving survivin, that are targeted by tumor viruses can lead to rapid and rational identification of drug candidates for treating virus-induced cancers.
The most frequent cause of α1-antitrypsin (here referred to as AT) deficiency is homozygosity for the AT-Z allele, which encodes AT-Z. Such individuals are at increased risk for liver disease due to the accumulation of aggregation-prone AT-Z in the endoplasmic reticulum of hepatocytes. However, the penetrance and severity of liver dysfunction in AT deficiency is variable, indicating that unknown genetic and environmental factors contribute to its occurrence. There is evidence that the rate of AT-Z degradation may be one such contributing factor. Through the use of several AT-Z model systems, it is now becoming appreciated that AT-Z can be degraded through at least two independent pathways. One model system that has contributed significantly to our understanding of the AT-Z disposal pathway is the yeast, Saccharomyces cerevisiae.
antitrypsin; endoplasmic reticulum–associated degradation; autophagy; yeast
Endoplasmic reticulum (ER) stress induces an mRNA decay pathway termed regulated Ire-dependent decay (RIDD). This study shows that in Drosophila cells, ER association is sufficient for targeting mRNAs to RIDD and most membrane-associated mRNAs are degraded more rapidly during ER stress. It is also reported that a small number of mRNAs are specifically protected from this default pathway.
Ire1 is an endoplasmic reticulum (ER) transmembrane protein that senses disturbances in protein folding homeostasis and contributes to a multifaceted response to stress. The nuclease activity of Ire1, in addition to splicing the mRNA encoding the transcription factor Xbp1, mediates mRNA degradation in response to ER stress through a pathway termed regulated Ire1-dependent decay (RIDD). We previously showed that ER targeting of substrates is necessary for RIDD; in this paper, we show that ER localization is also sufficient to induce decay in a normally unaffected mRNA. Using microarrays, we also measured relative mRNA degradation in the presence and absence of ER stress in Drosophila S2 cells, and determined mRNA membrane association using detergent fractionation. The vast majority of mRNAs that were strongly associated with the ER were degraded faster during ER stress in an Ire1-dependent manner, suggesting that RIDD is the default pathway for ER-localized mRNAs during stress. We also show that the mRNA encoding plexin A remains highly polysome associated during stress and escapes degradation by RIDD, and that its 5′ untranslated region can protect a strong RIDD target from degradation. These results suggest that while translation is generally attenuated during ER stress, continued translation of certain messages can protect them from degradation by RIDD.
This study describes new yeast expression systems for each subunit of the heterotrimeric epithelial sodium channel (ENaC). We found that a significant amount of each subunit resides in the ER and is destroyed via ERAD. We also found that the chaperone requirements for ENaC subunit degradation were unlike any other ERAD substrate examined.
The epithelial sodium channel (ENaC) is composed of a single copy of an α-, β-, and γ-subunit and plays an essential role in water and salt balance. Because ENaC assembles inefficiently after its insertion into the ER, a substantial percentage of each subunit is targeted for ER-associated degradation (ERAD). To define how the ENaC subunits are selected for degradation, we developed novel yeast expression systems for each ENaC subunit. Data from this analysis suggested that ENaC subunits display folding defects in more than one compartment and that subunit turnover might require a unique group of factors. Consistent with this hypothesis, yeast lacking the lumenal Hsp40s, Jem1 and Scj1, exhibited defects in ENaC degradation, whereas BiP function was dispensable. We also discovered that Jem1 and Scj1 assist in ENaC ubiquitination, and overexpression of ERdj3 and ERdj4, two lumenal mammalian Hsp40s, increased the proteasome-mediated degradation of ENaC in vertebrate cells. Our data indicate that Hsp40s can act independently of Hsp70 to select substrates for ERAD.
Agonist-induced glucocorticoid receptor [GR] transport from the cytoplasm to the nucleus was used as a model to identify dynein-mediated cargo transport inhibitors. Cell-based screening of the library of pharmacologically active compound (LOPAC)-1280 collection identified several small molecules that stalled the agonist-induced transport of GR-green fluorescent protein (GFP) in a concentration-dependent manner. Fluorescent images of microtubule organization, nuclear DNA staining, expression of GR-GFP, and its subcellular distribution were inspected and quantified by image analysis to evaluate the impact of compounds on cell morphology, toxicity, and GR transport. Given the complexity of the multi-protein complex involved in dynein-mediated cargo transport and the variety of potential mechanisms for interruption of that process, we therefore developed and validated a panel of biochemical assays to investigate some of the more likely intracellular target(s) of the GR transport inhibitors. Although the apomorphine enantiomers exhibited the most potency toward the ATPase activities of cytoplasmic dynein, myosin, and the heat-shock proteins (HSPs), their apparent lack of specificity made them unattractive for further study in our quest. Other molecules appeared to be nonspecific inhibitors that targeted reactive cysteines of proteins. Ideally, specific retrograde transport inhibitors would either target dynein itself or one of the other important proteins associated with the transport process. Although the hits from the cell-based screen of the LOPAC-1280 collection did not exhibit this desired profile, this screening platform provided a promising phenotypic system for the discovery of dynein/HSP modulators.
Secretory and membrane proteins that fail to fold in the endoplasmic reticulum (ER) are retained and may be sorted for ER-associated degradation (ERAD). During ERAD, ER-associated components such as molecular chaperones and lectins recognize folding intermediates and specific oligosaccharyl modifications on ERAD substrates. Substrates selected for ERAD are then targeted for ubiquitin- and proteasome-mediated degradation. Because the catalytic steps of the ubiquitin–proteasome system reside in the cytoplasm, soluble ERAD substrates that reside in the ER lumen must be retrotranslocated back to the cytoplasm prior to degradation. In contrast, it has been less clear how polytopic, integral membrane substrates are delivered to enzymes required for ubiquitin conjugation and to the proteasome. In this review, we discuss recent studies addressing how ERAD substrates are recognized, ubiquitinated and delivered to the proteasome and then survey current views of how soluble and integral membrane substrates may be retrotranslocated.
degradation; ER; glycosylation; molecular chaperone; proteasome; proteolysis; transport; ubiquitin
Endoplasmic reticulum-associated degradation (ERAD) is a process that clears the early secretory pathway of misfolded proteins. Though ERAD is of basic biological importance, the clinical importance of this pathway is emphasized by the fact that mutations that render a protein subject to the ERAD quality control pathway underlie the cause of several diseases. The yeast, Saccharomyces cerevisiae, is a valuable and frequently used model system to study biological processes, such as ERAD, as it is a relatively simple model system for which numerous biochemical and genetic tools are available. In addition, the ERAD system is highly conserved between yeast and man. In this chapter, we describe two methods for the analysis of model substrates that undergo catabolism via the ERAD pathway using S. cerevisiae. In particular, we will describe non-radioactive degradation assays and the analysis of substrate ubiquitylation in vivo with or without the use of ubiquitin overexpression systems. We also describe technical hurdles, which we have encountered in our research, and highlight remedies to overcome them.
Yeast; ER-associated degradation; Ubiquitin; Proteasome; Cycloheximide chase
Hsp110s are divergent relatives of Hsp70 chaperones that hydrolyze ATP. Hsp110s serve as Hsp70 nucleotide exchange factors and act directly to maintain polypeptide solubility. To date, the impact of peptide binding on Hsp110 ATPase activity is unknown and an Hsp110/peptide affinity has not been measured. We now report on a peptide that binds to the yeast Hsp110, Sse1p, with a KD of ~2 nM. Surprisingly, the binding of this peptide fails to stimulate Sse1p ATP hydrolysis. Moreover, an Hsp70-binding peptide is unable to associate with Sse1p, suggesting that Hsp70s and Hsp110s possess partially distinct peptide recognition motifs.
Hsp70; molecular chaperone; nucleotide exchange factor; fluorescence; ATPase
An increasing body of data links endoplasmic reticulum (ER) function to autophagy. Not surprisingly, then, some aberrant proteins in the ER can be destroyed either via ER associated degradation (ERAD), which is proteasome-mediated, or via autophagy. One such substrate is the “Z” variant of the alpha-1 protease inhibitor (A1Pi), variably known as A1Pi-Z or AT-Z (“anti-trypsin, Z variant”). The wild type protein is primarily synthesized in the liver and is secreted. In contrast, AT-Z, like other ERAD substrates, is retro-translocated from the ER and delivered to the proteasome. However, AT-Z can form high molecular weight polymers that are degraded via autophagy, and cells that accumulate AT-Z polymers ultimately succumb, which leads to liver disease. Therefore, identifying genes that have an impact AT-Z turnover represents an active area of research. To this end, a yeast expression system for AT-Z has proven valuable. For example, a recent study using this system indicates that the activity of a proteasome assembly chaperone (PAC) is critical for maximal AT-Z turnover, which suggests a new role for PACs. Because PACs are conserved, it will be critical to analyze whether these dedicated chaperones are implicated in other diseases associated with ERAD and autophagy.
anti-trypsin; A1Pi; proteasome; chaperone; proteasome assembly chaperone; PAC; yeast; unfolded protein response; liver disease; apoptosis
It remains unclear how misfolded membrane proteins are selected and destroyed during endoplasmic reticulum associated degradation (ERAD). For example, chaperones are thought to solubilize aggregation-prone motifs, and some data suggest that these proteins are degraded at the ER. To better define how membrane proteins are destroyed, the ERAD of Ste6p*, a twelve transmembrane protein, was reconstituted. We found that specific Hsp70/40s act before ubiquitination and facilitate Ste6p* association with an E3 ubiquitin ligase, suggesting an active role for chaperones. Furthermore, polyubiquitination was a prerequisite for retro-translocation, which required the Cdc48 complex and ATP. Surprisingly, the substrate was soluble, and extraction was independent of a ubiquitin chain extension enzyme (Ufd2p). However, Ufd2p increased the degree of ubiquitination and facilitated degradation. These data indicate that polytopic membrane proteins can be extracted from the ER, and define the point of action of chaperones and the requirement for Ufd2p during membrane protein quality control.
molecular chaperone; Hsp70; ubiquitination; Cdc48/p97; Ufd2; proteasome
Most proteins in the secretory pathway are translated, folded, and subjected to quality control at the endoplasmic reticulum (ER). These processes must be flexible enough to process diverse protein conformations, yet specific enough to recognize when a protein should be degraded. Molecular chaperones are responsible for this decision making process. ER associated chaperones assist in polypeptide translocation, protein folding, and ER associated degradation (ERAD). Nevertheless, we are only beginning to understand how chaperones function, how they are recruited to specific substrates and assist in folding/degradation, and how unique chaperone classes make quality control “decisions.”
heat shock proteins; ERAD; proteasome; lectin; degradation
Secreted proteins that fail to achieve their native conformations, such as cystic fibrosis transmembrane conductance regulator (CFTR) and particularly the ΔF508-CFTR variant can be selected for endoplasmic reticulum (ER)-associated degradation (ERAD) by molecular chaperones. Because the message corresponding to HSP26, which encodes a small heat-shock protein (sHsp) in yeast was up-regulated in response to CFTR expression, we examined the impact of sHsps on ERAD. First, we observed that CFTR was completely stabilized in cells lacking two partially redundant sHsps, Hsp26p and Hsp42p. Interestingly, the ERAD of a soluble and a related integral membrane protein were unaffected in yeast deleted for the genes encoding these sHsps, and CFTR polyubiquitination was also unaltered, suggesting that Hsp26p/Hsp42p are not essential for polyubiquitination. Next, we discovered that ΔF508-CFTR degradation was enhanced when a mammalian sHsp, αA-crystallin, was overexpressed in human embryonic kidney 293 cells, but wild-type CFTR biogenesis was unchanged. Because αA-crystallin interacted preferentially with ΔF508-CFTR and because purified αA-crystallin suppressed the aggregation of the first nucleotide-binding domain of CFTR, we suggest that sHsps maintain the solubility of ΔF508-CFTR during the ERAD of this polypeptide.
The evolution of eukaryotes was accompanied by an increased need for intracellular communication and cellular specialization. Thus, a more complex collection of secreted and membrane proteins had to be synthesized, modified, and folded. The endoplasmic reticulum (ER) thereby became equipped with devoted enzymes and associated factors that both catalyze the production of secreted proteins and remove damaged proteins. A means to modify ER function to accommodate and destroy misfolded proteins also evolved. Not surprisingly, a growing number of human diseases are linked to various facets of ER function. Each of these topics will be discussed in this article, with an emphasis on recent reports in the literature that employed diverse models.
Yos9p is involved in ER-associated degradation (ERAD) of misfolded glycoproteins. This study shows that Yos9p is required for ER retention of ERAD substrates by targeting them to the Hrd1p E3 ligase. This ER retention is independent of the glycan degradation signal on substrates and is separable from the later degradation step.
The endoplasmic reticulum (ER) has an elaborate quality control system, which retains misfolded proteins and targets them to ER-associated protein degradation (ERAD). To analyze sorting between ER retention and ER exit to the secretory pathway, we constructed fusion proteins containing both folded carboxypeptidase Y (CPY) and misfolded mutant CPY (CPY*) units. Although the luminal Hsp70 chaperone BiP interacts with the fusion proteins containing CPY* with similar efficiency, a lectin-like ERAD factor Yos9p binds to them with different efficiency. Correlation between efficiency of Yos9p interactions and ERAD of these fusion proteins indicates that Yos9p but not BiP functions in the retention of misfolded proteins for ERAD. Yos9p targets a CPY*-containing ERAD substrate to Hrd1p E3 ligase, thereby causing ER retention of the misfolded protein. This ER retention is independent of the glycan degradation signal on the misfolded protein and operates even when proteasomal degradation is inhibited. These results collectively indicate that Yos9p and Hrd1p mediate ER retention of misfolded proteins in the early stage of ERAD, which constitutes a process separable from the later degradation step.
Sss1p, an essential component of the heterotrimeric Sec61 complex in the endoplasmic reticulum (ER) #, is a tail-anchored protein whose precise mechanism of action is largely unknown. Tail-anchored proteins are involved in many cellular processes and are characterized by a single transmembrane sequence (TMS) at or near the carboxyl-terminus. The Sec61 complex is the molecular machine through which secretory and membrane proteins translocate into and across the ER membrane. To understand the function of the tail-anchor of Sss1p, we introduced mutations into the tail-anchor sequence and analyzed the resulting yeast phenotypes. Point mutations in the C-terminal hydrophobic core of the tail-anchor of Sss1p were identified that allowed Sss1p assembly into Sec61 complexes but resulted in diminished growth, defects in co- and post-translational translocation, diminished ribosome binding to Sec61 complexes, reduced stability of both heterotrimeric Sec61 and heptameric Sec complexes, and a complete breakdown of ER structure. The underlying defect caused by the mutations involves loss of a stabilizing function of the Sss1p tail-anchor sequence for both the heterotrimeric Sec61 and the heptameric Sec complexes. These data indicate that by stabilizing multiprotein membrane complexes, the hydrophobic core of a tail-anchor sequence can be more than a simple membrane anchor.
tail-anchor proteins; translocon; endoplasmic reticulum; membrane protein complexes; Sec61; yeast
Heat shock protein 70 (Hsp70) is a chaperone protein that helps protect against cellular stress, a function that may be co-opted to fight human diseases. In particular, the upregulation of Hsp70 can suppress the neurotoxicity of misfolded proteins, suggesting possible therapeutic strategies in neurodegenerative diseases. Alternatively, in cancer cells where high levels of Hsp70 inhibit both intrinsic and extrinsic apoptotic pathways, a reduction in Hsp70 levels may induce apoptosis. To evaluate and identify, in a single assay format, small molecules that induce or inhibit endogenous Hsp70, we have designed and optimized a microtiter assay that relies on whole-cell immunodetection of Hsp70. The assay utilizes a minimal number of neuronal or cancer cells, yet is sufficiently sensitive and reproducible to permit quantitative determinations. We further validated the assay using a panel of Hsp70 modulators. In conclusion, we have developed an assay that is fast, robust, and cost efficient. As such, it can be implemented in most research laboratories. The assay should greatly improve the speed at which novel Hsp70 inducers and inhibitors of expression can be identified and evaluated.
IRE1 signaling robustly promoted P23H rhodopsin degradation by both proteasome and lysosome. When one degradation pathway was blocked, IRE1 signaling still promoted misfolded rhodopsin degradation using the remaining pathway. IRE1 signaling reduced levels of other misfolded rhodopsins, with lesser effects on misfolded CFTR.
Endoplasmic reticulum (ER) is responsible for folding of secreted and membrane proteins in eukaryotic cells. Disruption of ER protein folding leads to ER stress. Chronic ER stress can cause cell death and is proposed to underlie the pathogenesis of many human diseases. Inositol-requiring enzyme 1 (IRE1) directs a key unfolded protein response signaling pathway that controls the fidelity of ER protein folding. IRE1 signaling may be particularly helpful in preventing chronic ER stress and cell injury by alleviating protein misfolding in the ER. To examine this, we used a chemical-genetic approach to selectively activate IRE1 in mammalian cells and tested how artificial IRE1 signaling affected the fate of misfolded P23H rhodopsin linked to photoreceptor cell death. We found that IRE1 signaling robustly promoted the degradation of misfolded P23H rhodopsin without affecting its wild-type counterpart. We also found that IRE1 used both proteasomal and lysosomal degradation pathways to remove P23H rhodopsin. Surprisingly, when one degradation pathway was compromised, IRE1 signaling could still promote misfolded rhodopsin degradation using the remaining pathway. Last, we showed that IRE1 signaling also reduced levels of several other misfolded rhodopsins with lesser effects on misfolded cystic fibrosis transmembrane conductance regulator. Our findings reveal the diversity of proteolytic mechanisms used by IRE1 to eliminate misfolded rhodopsin.
Protein disulfide isomerases (PDIs) are conserved chaperone-like proteins that play an essential role during protein folding and in some cases during degradation. Substrate-specific effects of PDI family members occur during the ER-associated degradation of diverse substrates in yeast and mammalian cells.
ER-associated degradation (ERAD) rids the early secretory pathway of misfolded or misprocessed proteins. Some members of the protein disulfide isomerase (PDI) family appear to facilitate ERAD substrate selection and retrotranslocation, but a thorough characterization of PDIs during the degradation of diverse substrates has not been undertaken, in part because there are 20 PDI family members in mammals. PDIs can also exhibit disulfide redox, isomerization, and/or chaperone activity, but which of these activities is required for the ERAD of different substrate classes is unknown. We therefore examined the fates of unique substrates in yeast, which expresses five PDIs. Through the use of a yeast expression system for apolipoprotein B (ApoB), which is disulfide rich, we discovered that Pdi1 interacts with ApoB and facilitates degradation through its chaperone activity. In contrast, Pdi1's redox activity was required for the ERAD of CPY* (a misfolded version of carboxypeptidase Y that has five disulfide bonds). The ERAD of another substrate, the alpha subunit of the epithelial sodium channel, was Pdi1 independent. Distinct effects of mammalian PDI homologues on ApoB degradation were then observed in hepatic cells. These data indicate that PDIs contribute to the ERAD of proteins through different mechanisms and that PDI diversity is critical to recognize the spectrum of potential ERAD substrates.
The CFTR Folding Consortium (CFC) was formed in 2004 under the auspices of the Cystic Fibrosis Foundation and its drug discovery and development affiliate, CFF Therapeutics. A primary goal of the CFC is the development and distribution of reagents and assay methods designed to better understand the mechanistic basis of mutant CFTR misfolding and to identify targets whose manipulation may correct CFTR folding defects. As such, reagents available from the CFC primarily target wild-type CFTR NBD1 and its common variant, F508del, and they include antibodies, cell lines, constructs, and proteins. These reagents are summarized here, and two protocols are described for the detection of cell surface CFTR: (a) an assay of the density of expressed HA-tagged CFTR by ELISA and (b) the generation and use of an antibody to CFTR’s first extracellular loop for the detection of endogenous CFTR. Finally, we highlight a systematic collection of assays, the CFC Roadmap, which is being used to assess the cellular locus and mechanism of mutant CFTR correction. The Roadmap queries CFTR structure–function relations at levels ranging from purified protein to well-differentiated human airway primary cultures.
Protein folding; protein degradation; antibody generation; cell surface protein detection; research consortium; www.cftrfolding.org
Heat shock protein 70 (Hsp70) and heat shock protein 40 (Hsp40) function as molecular chaperones during the folding and trafficking of proteins within most cell types. However, the Hsp70–Hsp40 chaperone partnerships within the malaria parasite, Plasmodium falciparum, have not been elucidated. Only one of the 43 P. falciparum Hsp40s is predicted to be a cytosolic, canonical Hsp40 (termed PfHsp40) capable of interacting with the major cytosolic P. falciparum-encoded Hsp70, PfHsp70. Consistent with this hypothesis, we found that PfHsp40 is upregulated under heat shock conditions in a similar pattern to PfHsp70. In addition, PfHsp70 and PfHsp40 reside mainly in the parasite cytosol, as assessed using indirect immunofluorescence microscopy. Recombinant PfHsp40 stimulated the ATP hydrolytic rates of both PfHsp70 and human Hsp70 similar to other canonical Hsp40s of yeast (Ydj1) and human (Hdj2) origin. In contrast, the Hsp40-stimulated plasmodial and human Hsp70 ATPase activities were differentially inhibited in the presence of pyrimidinone-based small molecule modulators. To further probe the chaperone properties of PfHsp40, protein aggregation suppression assays were conducted. PfHsp40 alone suppressed protein aggregation, and cooperated with PfHsp70 to suppress aggregation. Together, these data represent the first cellular and biochemical evidence for a PfHsp70–PfHsp40 partnership in the malaria parasite, and furthermore that the plasmodial and human Hsp70–Hsp40 chaperones possess unique attributes that are differentially modulated by small molecules.
Electronic supplementary material
The online version of this article (doi:10.1007/s12192-010-0250-6) contains supplementary material, which is available to authorized users.
Aggregation; ATPase; Codon harmonisation; Heat shock protein; Malaria; Molecular chaperone