Highly efficient degradation of acetaminophen via nano zero-valent iron biochar with periodate system at low temperature.

The development of more efficient advanced oxidation systems for serving various advanced treatment of wastewater is quite necessary and urgent. In this study, a nano-zero valent iron/periodate (nZVI-BC/PI) advanced oxidation system has been constructed, achieving a rapid degradation of acetaminophen (ACT, 1 mg/L) within 1 min (100 % at pH = 11) at low temperature (5℃). This system shows a great degradation in a wide range of pH (1 âˆ¼ 11), improving the pH limitation of PI oxidation system. During the reaction process, ·OH as the main active species collaborate with 1O2, Fe (IV), ·O2- and electron transfer to degrade ACT. In this system, iron ion leaching is low (0.019 mg/L), ACT was effectively degraded (74.36 %∼97.32 %) under different water, moreover, the material has an expected recyclability. The research provides a significant guidance for the advanced treatment of wastewater especially in cold regions.

Thallium-mediated NO signaling induced lipid accumulation in microalgae and its role in heavy metal bioremediation.

Thallium (Tl+) is a trace metal with extreme toxicity and is highly soluble in water, posing a great risk to ecological and human safety. This work aimed to investigate the role played by Tl+ in regulating lipid accumulation in microalgae and the removal efficiency of Tl+. The effect of Tl+ on the cell growth, lipid production and Tl+ removal efficiency of Parachlorella kessleri R-3 was studied. Low concentrations of Tl+ had no significant effect on the biomass of microalgae. When the Tl+ concentration exceeded 5 µg L-1, the biomass of microalgae showed significant decrease. The highest lipid content of 63.65% and lipid productivity of 334.55 mg L-1 d-1 were obtained in microalgae treated with 10 and 5 µg L-1 Tl+, respectively. Microalgae can efficiently remove Tl+ and the Tl+ removal efficiency can reach 100% at Tl+ concentrations of 0-25 µg L-1. The maximum nitric oxide (NO) level of 470.48 fluorescence intensity (1 × 106 cells)-1 and glutathione (GSH) content of 343.51 nmol g-1 (fresh alga) were obtained under 5 µg L-1 Tl+ stress conditions. Furthermore, the exogenous donor sodium nitroprusside (SNP) supplemented with NO was induced in microalgae to obtain a high lipid content (59.99%), lipid productivity (397.99 mg L-1 d-1) and GSH content (430.22 nmol g-1 (fresh alga)). The corresponding analysis results indicated that NO could participate in the signal transduction pathway through modulation of reactive oxygen species (ROS) signaling to activate the antioxidant system by increasing the GSH content to eliminate oxidative damage induced by Tl+ stress. In addition, NO regulation of ROS signaling may enhance transcription factors associated with lipid synthesis, which stimulates the expression of genes related to lipid synthesis, leading to increased lipid biosynthesis in microalgae. Moreover, it was found that the change in Tl+ had little effect on the fatty acid components and biodiesel properties. This study showed that Tl+ stress can promote lipid accumulation in microalgae for biodiesel production and simultaneously effectively remove Tl+, which provided evidence that NO was involved in signal transduction and antioxidant defense, and improved the understanding of the interrelation between NO and ROS to regulate lipid accumulation in microalgae.

Legionnaires' Disease in China Caused by Legionella pneumophila Corby.

Legionella pneumophila is an intracellular pathogen causing pneumonia in humans. In February 2022, Legionnaires' disease caused by L. pneumophila strain Corby in a patient with lung adenocarcinoma was identified for the first time in China. This paper includes the case report and phenotypic and genomic analysis of the Corby (ICDC) strain. Its biological characteristics were evaluated by antibiotic sensitivity testing and cytology experiments, and genomic analysis was performed to understand its genetic evolution. The patient's clinical manifestations included cough, fever, pulmonary infiltration, and significantly decreased activity endurance. After empirical antimicrobial therapy, infection indicators decreased. The Corby (ICDC) strain was susceptible to nine antibiotics and exhibited strong intracellular proliferation ability. A phylogenetic tree showed that the Corby (ICDC) strain was closely related to the Corby strain, but under the pressure of a complex environment, its genome had undergone more rearrangement and inversion. The type IF CRISPR-Cas system was identified in its genome, and spacer analysis indicated that it had been invaded by several foreign plasmids, bacteria, and viruses during evolution. Legionnaires' disease caused by L. pneumophila strain Corby may be ignored in China, and it is urgent to improve long-term monitoring and investigation of aquatic environments and patients with respiratory infections to prevent a large-scale outbreak of Legionnaires' disease.

Synergistic effect of hydrogen and nanoscale zero-valent iron on ex-situ biogas upgrading and acetate recovery.

Hydrogen (H2) assisted ex-situ biogas upgrading and liquid chemicals production can augment the fossil fuel-dominated energy market, and alleviate CO2-induced global warming. Recent investigations confirmed that nanoscale zero-valent iron (nZVI) enabled the enhancement of anaerobic digestion for biogas production. However, little is known about the effect of nZVI on the downstream ex-situ biogas upgrading. Herein, different levels (0 mg L-1, 100 mg L-1, 200 mg L-1, 500 mg L-1, 1000 mg L-1, 2000 mg L-1) of nZVI were added for H2-assisted ex-situ biogas upgrading, to study whether nZVI could impact the biomethane purity and acetate yield for the first time. Results showed that all tested nZVI levels were favorable for biogas upgrading in the presence of H2, the highest biomethane content (94.1 %, v/v), the CO2 utilization ratio (95.9 %), and acetate yield (19.4 mmol L-1) were achieved at 500 mg L-1 nZVI, respectively. Further analysis indicated that increased biogas upgrading efficiency was related to an increase in extracellular polymeric substances, which ensures the microbial activity and stability of the ex-situ biogas upgrading. Microbial community characterization showed that the Petrimonas, Romboutsia, Acidaminococcus, and Clostridium predominated the microbiome during biogas upgrading at 500 mg L-1 nZVI with H2 supply. These results suggested that nZVI and H2 contributed jointly to promoting the bioconversion of CO2 in biogas to acetate. The findings could be helpful for paving a new way for efficient simultaneous ex-situ biogas upgrading and liquid chemicals recovery.

Role of residue cornstalk derived biochar for the enhanced bio-hydrogen production via simultaneous saccharification and fermentation of cornstalk.

Biochar derived from residue cornstalk left after anaerobic bio-hydrogen production (RCA-biochar) was confirmed to enhance bio-hydrogen production from cornstalk hydrolysate. However, the role of RCA-biochar in simultaneous saccharification and fermentation (SSF) during bio-hydrogen production from cornstalk has not yet been revealed. This study therefore aims to fill this knowledge gap. It was observed that with the increase in RCA-biochar concentration from 0 g/L to 10.0 g/L, the maximal cumulative SSF bio-hydrogen yield varied from 24.3 ± 1.1 mL/g-substrate to 154.3 ± 3.6 mL/g substrate under varying pH values - 5.5, 6.0, 6.5, 7.0. The increasing bio-hydrogen production was observed to correlate with both RCA-biochar level and initial pH. Batch tests confirmed that the initial pH had an obvious effect an saccharification, while RCA-biochar affected anaerobic fermentation a lot. The findings revealed the role of previously unrecognized RCA-biochar in SSF bio-hydrogen production from cornstalk, which can provide an alternative approach for lignocellulosic bio-hydrogen production.

DNAJB12 and Hsp70 triage arrested intermediates of N1303K-CFTR for endoplasmic reticulum-associated autophagy.

The transmembrane Hsp40 DNAJB12 and cytosolic Hsp70 cooperate on the endoplasmic reticulum's (ER) cytoplasmic face to facilitate the triage of nascent polytopic membrane proteins for folding versus degradation. N1303K is a common mutation that causes misfolding of the ion channel CFTR, but unlike F508del-CFTR, biogenic and functional defects in N1303K-CFTR are resistant to correction by folding modulators. N1303K is reported to arrest CFTR folding at a late stage after partial assembly of its N-terminal domains. N1303K-CFTR intermediates are clients of JB12-Hsp70 complexes, maintained in a detergent-soluble state, and have a relatively long 3-h half-life. ER-associated degradation (ERAD)-resistant pools of N1303K-CFTR are concentrated in ER tubules that associate with autophagy initiation sites containing WIPI1, FlP200, and LC3. Destabilization of N1303K-CFTR or depletion of JB12 prevents entry of N1303K-CFTR into the membranes of ER-connected phagophores and traffic to autolysosomes. In contrast, the stabilization of intermediates with the modulator VX-809 promotes the association of N1303K-CFTR with autophagy initiation machinery. N1303K-CFTR is excluded from the ER-exit sites, and its passage from the ER to autolysosomes does not require ER-phagy receptors. DNAJB12 operates in biosynthetically active ER microdomains to triage membrane protein intermediates in a conformation-specific manner for secretion versus degradation via ERAD or selective-ER-associated autophagy.

Residue cornstalk derived biochar promotes direct bio-hydrogen production from anaerobic fermentation of cornstalk.

In this study, an innovative approach was proposed based on the implement of biochar derived from residue cornstalk left after anaerobic bio-hydrogen production (RCA-biochar) to improve direct bio-hydrogen production from anaerobic fermentation of cornstalk. The bio-hydrogen production potential and maximum bio-hydrogen production rate increased from 156.2 to 286.1 mL H2/g substrate and 3.5 to 5.7 mL H2/g substrate/h, respectively, following the added RCA-biochar increased from 2.5 to 15.0 g/L. Cornstalk chemical component analysis showed the cellulose and hemicellulose content decreased by 17.9-33.7% and 14.4-25.2%, and lignin content increased by 20.3-42.8%, respectively, after 96 h anaerobic fermentation with RCA-biochar 2.5-15.0 g/L. Further analyses revealed that RCA-biochar not only provided more specific surface area for hydrogen-producing bacteria attachment, but also promoted the cellulolytic enzyme activity, thereby resulted in increased substrate conversion to bio-hydrogen.The findings obtained in this study may provide supports for effective and sustainable lignocellulosic bio-hydrogen production in the future.

Sheng-Jiang Powder Ameliorates High Fat Diet Induced Nonalcoholic Fatty Liver Disease via Inhibiting Activation of Akt/mTOR/S6 Pathway in Rats.

BACKGROUND AND AIMS: Nonalcoholic fatty liver disease (NAFLD) is an alarming public health problem that directly contributes to increased prevalence of liver cirrhosis and hepatic cell cancer, but without any specific pharmacological option. Sheng-jiang powder (SJP), an empirical Chinese medicine formula to treat NAFLD, showed great efficacy but the specific mechanisms have never been reported. Therefore, we performed this study to explore the effect of SJP on NAFLD and the potential mechanism. METHODS: NAFLD was induced by high fat diet (HFD) feeding. Rats were treated with SJP/normal saline daily for 10 weeks and all rats were euthanized after 12 weeks' feeding. Liver tissue samples were obtained for biochemistry test and pathological evaluation. Additionally, oleic acid induced LO2 cells were employed to simulate a cell model of NAFLD. Cells were subjected to western blotting for Akt, mTOR, S6, SREBP1-c, and FASN detection after coincubated with SJP, LY294002 (a selective PI3K inhibitor), or the combination for 24h. RESULTS: HFD significantly induced hepatic steatosis. Plenty of lipid droplets were observed under transmission electron microscope. The ultrastructure of liver cells showed distinct changes with obvious endoplasmic reticulum expansion, mitochondrial contraction, and cell matrix solidification. Although no difference was detected in serum hepatic enzymes and tissue proinflammatory cytokines, the tissue level of SOD and GSH-px was much lower and the pathologic injuries were much severe in HFD feeding rats. However, SJP treatment significantly attenuated the ultrastructure changes and protected rat liver against inflammatory injury. Abundant of lipid droplets and high expression of pAkt, pmTOR, pS6, and FASN were observed in oleic acid treated LO2 cells, while these changes were restored by SJP treatment. CONCLUSIONS: SJP is efficient in attenuating HFD induced NAFLD in rats and this effect might be partly related to the inhibition of Akt/mTOR/S6 pathway.

Endoplasmic reticulum stress-induced degradation of DNAJB12 stimulates BOK accumulation and primes cancer cells for apoptosis.

DNAJB12 (JB12) is an endoplasmic reticulum (ER)-associated Hsp40 family protein that recruits Hsp70 to the ER surface to coordinate the function of ER-associated and cytosolic chaperone systems in protein quality control. Hsp70 is stress-inducible, but paradoxically, we report here that JB12 was degraded by the proteasome during severe ER stress. Destabilized JB12 was degraded by ER-associated degradation complexes that contained HERP, Sel1L, and gp78. JB12 was the only ER-associated chaperone that was destabilized by reductive stress. JB12 knockdown by siRNA led to the induction of caspase processing but not the unfolded protein response. ER stress-induced apoptosis is regulated by the highly labile and ER-associated BCL-2 family member BOK, which is controlled at the level of protein stability by ER-associated degradation components. We found that JB12 was required in human hepatoma cell line 7 (Huh-7) liver cancer cells to maintain BOK at low levels, and BOK was detected in complexes with JB12 and gp78. Depletion of JB12 during reductive stress or by shRNA from Huh-7 cells was associated with accumulation of BOK and activation of Caspase 3, 7, and 9. The absence of JB12 sensitized Huh-7 to death caused by proteotoxic agents and the proapoptotic chemotherapeutic LCL-161. In summary, JB12 is a stress-sensitive Hsp40 whose degradation during severe ER stress provides a mechanism to promote BOK accumulation and induction of apoptosis.

Pharmacokinetics and pharmacodynamics of Da-Cheng-Qi decoction in the liver of rats with severe acute pancreatitis.

AIM: To explore the pharmacokinetics and pharmacodynamics of Da-Cheng-Qi decoction (DCQD) in the liver of rats with severe acute pancreatitis (SAP) based on an herbal recipe tissue pharmacology hypothesis. METHODS: Healthy male Sprague-Dawley rats were randomly divided into a sham operation group (SOG); a model group (MG); and low-, median- and high-dose treatment groups (LDG, MDG, and HDG, respectively). Different dosages (6, 12 and 24 g/kg for the LDG, MDG, and HDG, respectively) of DCQD were administered to the rats with SAP. The tissue concentrations of aloe-emodin, rhein, emodin, chrysophanol, honokiol, rheo chrysophanol, magnolol, hesperidin, naringenin and naringin in the liver of the treated rats were detected by high-performance liquid chromatography tandem mass spectrometry. Alanine transaminase (ALT) and aspartate transaminase (AST) in serum, inflammatory mediators in the liver and pathological scores were evaluated. RESULTS: The major components of DCQD were detected in the liver, and their concentrations increased dose-dependently. The high dose of DCQD showed a maximal effect in ameliorating the pathological damages, decreasing the pro-inflammatory mediators tumor necrosis factor-α and interleukin (IL)-6 and increasing anti-inflammatory mediators IL-4 and IL-10 in the liver. The pathological scores in the pancreas for the MG were significantly higher than those for the SOG (P < 0.05). DCQD could reduce the pathological scores in the pancreas and liver of the rats with SAP, especially in the HDG. Compared to the SOG, the ALT and AST levels in serum were higher in the MG (P < 0.05), while there was no statistical difference in the MG and HDG. CONCLUSION: DCQD could alleviate liver damage by altering the inflammatory response in rats with SAP based on the liver distribution of its components.

Improvement of Quality of Nonanesthetic Colonoscopy by Preoperative Administration of Pinaverium Bromide.

BACKGROUND: Nonanesthetic colonoscopy is popular in clinical practice in China. However, intestinal spasms often result in a prolonged examination time, increased operating difficulties, decreased polyp detection rate, and failure to complete the procedure clinically. Therefore, exploring alternative approaches that can reduce the pain in patients during colonoscopy is of utmost importance, and finding the optimal preoperative administration to improve the quality of nonanesthetic colonoscopy is also necessary. This study aimed to investigate the effects of the prophylactic administration of pinaverium bromide before colonoscopy and the effects of pinaverium bromide alone at different time points or combined with scopolamine butylbromide. METHODS: A randomized controlled trial was performed on a cohort of 1000 patients who underwent colonoscopy in outpatient clinic of Wuhan Union Hospital. The patients were randomly assigned to the following groups: Group A, given oral pinaverium bromide (100 mg, three times a day) one day before examination combined with intramuscular injection of scopolamine butylbromide (20 mg) 10 min before colonoscopy; Group B0, given pinaverium bromide alone on the day of colonoscopy (100 mg, three times a day); Group B1, given pinaverium bromide alone (100 mg, three times a day) one day before colonoscopy; Group B2, given pinaverium bromide alone (100 mg, three times a day) two days before colonoscopy; and Group C, given scopolamine butylbromide alone (20 mg) before colonoscopy. The successful rate of colonoscopy, procedure time, degree of abdominal pain, and polyp detection rate were recorded and compared among all groups. RESULTS: The successful rate of colonoscopy in Group B1(82.0%) and Group B2(83.0%) was significantly higher than that in Group B0(62.0%, all P < 0.01). The time to reach the ileocecal region in Group B1and Group B2were lower than those in Group B0(all P < 0.05). However, no significant differences were observed in polyp detection rate between Group B1(24.0%) or Group B2(26.0%), and Group B0(22.4%, all P > 0.05). Furthermore, there were no significant differences in the various parameters examined between Group B1and Group B2(P > 0.05). The successful rate of colonoscopy in Group A (92.0%) was significantly higher than that in Group B1(82.0%) and Group C (80.0%; both P < 0.05). Moreover, the time for the colonoscope to reach the ileocecal region in Group A were markedly shorter as compared to those in Group B1 and Group C (P < 0.05). The polyp detection rate in Group A was 32.0%, significantly higher than that in Group B1(24.0%, P < 0.05) and Group C (24.2%, P < 0.05). CONCLUSION: Administration of pinaverium bromide alone one day before examination was beneficial to relieve symptoms of abdominal pain during nonanesthetic colonoscopy. In addition, therapeutic effects were improved when pinaverium bromide administration was combined with intramuscular injection of scopolamine butylbromide. Therefore, the combined use of pinaverium bromide with scopolamine butylbromide might have great application value to improve the quality of nonanesthetic colonoscopy in the preoperative preparation.

Bioaggregate of photo-fermentative bacteria for enhancing continuous hydrogen production in a sequencing batch photobioreactor.

Hydrogen recovery through solar-driven biomass conversion by photo-fermentative bacteria (PFB) has been regarded as a promising way for sustainable energy production. However, a considerable fraction of organic substrate was consumed for the growth of PFB as biocatalysts, furthermore, these PFB were continuously washed out from the photobioreactor in continuous operation because of their poor flocculation. In this work, PFB bioaggregate induced by L-cysteine was applied in a sequencing batch photobioreactor to enhance continuous hydrogen production and reduce biomass washout. The effects of the hydraulic retention time (HRT), influent concentration and light intensity on hydrogen production of the photobioreactor were investigated. The maximum hydrogen yield (3.35 mol H2/mol acetate) and production rate (1044 ml/l/d) were obtained at the HRT of 96 h, influent concentration of 3.84 g COD/l, and light intensity of 200 W/m(2). With excellent settling ability, biomass accumulated in the photobioreactor and reached 2.15 g/l under the optimum conditions. Structural analysis of bioaggregate showed that bacterial cells were covered and tightly linked together by extracellular polymeric substances, and formed a stable structure. Therefore, PFB bioaggregate induced by L-cysteine is an efficient strategy to improve biomass retention capacity of the photobioreactor and enhance hydrogen recovery efficiency from organic wastes.

Improved 4-chlorophenol dechlorination at biocathode in bioelectrochemical system using optimized modular cathode design with composite stainless steel and carbon-based materials.

This study developed and optimized a modular biocathode materials design in bioelectrochemical system (BES) using composite metal and carbon-based materials. The 4-chlorophenol (4-CP) dechlorination could be improved with such composite materials. Results showed that stainless steel basket (SSB) filled with graphite granules (GG) and carbon brush (CB) (SSB/GG/CB) was optimum for dechlorination, followed by SSB/CB and SSB/GG, with rate constant k of 0.0418 ± 0.0002, 0.0374 ± 0.0004, and 0.0239 ± 0.0002 h(-1), respectively. Electrochemical impedance spectroscopy (EIS) demonstrated that the composite materials with metal can benefit the electron transfer and decrease the charge transfer resistance to be 80.4 Ω in BES-SSB/GG/CB, much lower than that in BES-SSB (1674.3 Ω), BES-GG (387.3 Ω), and BES-CB (193.8 Ω). This modular cathode design would be scalable with successive modules for BES scale-up, and may offer useful information to guide the selection and design of BES materials towards dechlorination improvement in wastewater treatment.

Polyglutamine-rich suppressors of huntingtin toxicity act upstream of Hsp70 and Sti1 in spatial quality control of amyloid-like proteins.

Protein conformational maladies such as Huntington Disease are characterized by accumulation of intracellular and extracellular protein inclusions containing amyloid-like proteins. There is an inverse correlation between proteotoxicity and aggregation, so facilitated protein aggregation appears cytoprotective. To define mechanisms for protective protein aggregation, a screen for suppressors of nuclear huntingtin (Htt103Q) toxicity was conducted. Nuclear Htt103Q is highly toxic and less aggregation prone than its cytosolic form, so we identified suppressors of cytotoxicity caused by Htt103Q tagged with a nuclear localization signal (NLS). High copy suppressors of Htt103Q-NLS toxicity include the polyQ-domain containing proteins Nab3, Pop2, and Cbk1, and each suppresses Htt toxicity via a different mechanism. Htt103Q-NLS appears to inactivate the essential functions of Nab3 in RNA processing in the nucleus. Function of Pop2 and Cbk1 is not impaired by nuclear Htt103Q, as their respective polyQ-rich domains are sufficient to suppress Htt103Q toxicity. Pop2 is a subunit of an RNA processing complex and is localized throughout the cytoplasm. Expression of just the Pop2 polyQ domain and an adjacent proline-rich stretch is sufficient to suppress Htt103Q toxicity. The proline-rich domain in Pop2 resembles an aggresome targeting signal, so Pop2 may act in trans to positively impact spatial quality control of Htt103Q. Cbk1 accumulates in discrete perinuclear foci and overexpression of the Cbk1 polyQ domain concentrates diffuse Htt103Q into these foci, which correlates with suppression of Htt toxicity. Protective action of Pop2 and Cbk1 in spatial quality control is dependent upon the Hsp70 co-chaperone Sti1, which packages amyloid-like proteins into benign foci. Protein:protein interactions between Htt103Q and its intracellular neighbors lead to toxic and protective outcomes. A subset of polyQ-rich proteins buffer amyloid toxicity by funneling toxic aggregation intermediates to the Hsp70/Sti1 system for spatial organization into benign species.

Impact of probiotics on toll-like receptor 4 expression in an experimental model of ulcerative colitis.

Toll-like receptors (TLRs) are key components of the innate immune system which trigger antimicrobial host defense responses. This study aimed to investigate the impact of probiotics (Lactobacillus, Bifidobacterium) on the expression of TLR4 and tumor necrosis factor-alpha (TNF-α) in the colon mucosa of rat experimental ulcerative colitis model induced by trinitrobenzene sulfonic acid (TNBS)/ethanol and immune complexes. The gross and histological changes of the colonic mucosa were observed and assessed by the means-standard deviation and independent samples t-test. The protein expression levels of TLR4 and TNF-α were detected by using immunohistochemistry and Western blotting, respectively. It was revealed that there was visible infiltration of inflammatory cells, formation of crypt abscess, and the reduction of goblet cells in the colon tissue of experimental models. As compared with the control group, the levels of TLR4 and TNF-α protein were significantly increased in the model group (P<0.01 for both). No significant difference was found in the expression of TLR4 and TNF-α between the two-week probiotics treatment group and the model group (P>0.05), whereas significant reductions were shown in rats which were treated with probiotics for four weeks as compared with the model group (P<0.01). There was no significant difference between two probiotics-treated groups. Our results implied that probiotics were likely to play a key role in protecting ulcerative colitis by reducing the inflammatory factor TNF-α expression through inhibiting the TLR4 expression in the colon tissue of experimental models.

VX-809 corrects folding defects in cystic fibrosis transmembrane conductance regulator protein through action on membrane-spanning domain 1.

Cystic fibrosis (CF) is a fatal genetic disorder associated with defective hydration of lung airways due to the loss of chloride transport through the CF transmembrane conductance regulator protein (CFTR). CFTR contains two membrane-spanning domains (MSDs), two nucleotide-binding domains (NBDs), and a regulatory domain, and its channel assembly requires multiple interdomain contacts. The most common CF-causing mutation, F508del, occurs in NBD1 and results in misfolding and premature degradation of F508del-CFTR. VX-809 is an investigational CFTR corrector that partially restores CFTR function in people who are homozygous for F508del-CFTR. To identify the folding defect(s) in F508del-CFTR that must be repaired to treat CF, we explored the mechanism of VX-809 action. VX-809 stabilized an N-terminal domain in CFTR that contains only MSD1 and efficaciously restored function to CFTR forms that have missense mutations in MSD1. The action of VX-809 on MSD1 appears to suppress folding defects in F508del-CFTR by enhancing interactions among the NBD1, MSD1, and MSD2 domains. The ability of VX-809 to correct F508del-CFTR is enhanced when combined with mutations that improve F508del-NBD1 interaction with MSD2. These data suggest that the use of VX-809 in combination with an additional CFTR corrector that suppresses folding defects downstream of MSD1 may further enhance CFTR function in people with F508del-CFTR.

The Type II Hsp40 Sis1 cooperates with Hsp70 and the E3 ligase Ubr1 to promote degradation of terminally misfolded cytosolic protein.

Mechanisms for cooperation between the cytosolic Hsp70 system and the ubiquitin proteasome system during protein triage are not clear. Herein, we identify new mechanisms for selection of misfolded cytosolic proteins for degradation via defining functional interactions between specific cytosolic Hsp70/Hsp40 pairs and quality control ubiquitin ligases. These studies revolved around the use of S. cerevisiae to elucidate the degradation pathway of a terminally misfolded reporter protein, short-lived GFP (slGFP). The Type I Hsp40 Ydj1 acts with Hsp70 to suppress slGFP aggregation. In contrast, the Type II Hsp40 Sis1 is required for proteasomal degradation of slGFP. Sis1 and Hsp70 operate sequentially with the quality control E3 ubiquitin ligase Ubr1 to target slGFP for degradation. Compromise of Sis1 or Ubr1 function leads slGFP to accumulate in a Triton X-100-soluble state with slGFP degradation intermediates being concentrated into perinuclear and peripheral puncta. Interestingly, when Sis1 activity is low the slGFP that is concentrated into puncta can be liberated from puncta and subsequently degraded. Conversely, in the absence of Ubr1, slGFP and the puncta that contain slGFP are relatively stable. Ubr1 mediates proteasomal degradation of slGFP that is released from cytosolic protein handling centers. Pathways for proteasomal degradation of misfolded cytosolic proteins involve functional interplay between Type II Hsp40/Hsp70 chaperone pairs, PQC E3 ligases, and storage depots for misfolded proteins.

Reconstitution of CHIP E3 ubiquitin ligase activity.

CHIP, the carboxyl-terminus of Hsp70 interacting protein, is both an E3 ubiquitin ligase and an Hsp70 co-chaperone and is implicated in the degradation of cytosolic quality control and numerous disease substrates. CHIP has been shown to monitor the folding status of the CFTR protein, and we have successfully reconstituted this activity using a recombinant CFTR fragment consisting of the cytosolic NBD1 and R domains. We have found that efficient ubiquitination of substrates requires chaperone activity to either deliver the substrate to CHIP or to maintain the substrate in a ubiquitination-competent conformation. This chaperone activity can be provided by the Hsp70/Hsp40 molecular chaperone system as seen in the NBD1-R ubiquitination assay. Alternatively, heat treatment of CHIP can activate its own innate substrate-binding activity and allow for efficient ubiquitination of model substrates, such as denatured luciferase. Here, we describe methods for purifying the recombinant proteins necessary for in vitro reconstitution of CHIP ubiquitin ligase activity, as well as two methods used to monitor CHIP ligase activity. One method allows for the measurement of the Hsp70- and Hsp40-dependent CHIP activity while the other measures the Hsp40- and Hsp70-independent activity of heat-activated CHIP.

The endoplasmic reticulum-associated Hsp40 DNAJB12 and Hsc70 cooperate to facilitate RMA1 E3-dependent degradation of nascent CFTRDeltaF508.

Relative contributions of folding kinetics versus protein quality control (QC) activity in the partitioning of non-native proteins between life and death are not clear. Cystic fibrosis transmembrane conductance regulator (CFTR) biogenesis serves as an excellent model to study this question because folding of nascent CFTR is inefficient and deletion of F508 causes accumulation of CFTRΔF508 in a kinetically trapped, but foldable state. Herein, a novel endoplasmic reticulum (ER)-associated Hsp40, DNAJB12 (JB12) is demonstrated to play a role in control of CFTR folding efficiency. JB12 cooperates with cytosolic Hsc70 and the ubiquitin ligase RMA1 to target CFTR and CFTRΔF508 for degradation. Modest elevation of JB12 decreased nascent CFTR and CFTRΔF508 accumulation while increasing association of Hsc70 with ER forms of CFTR and the RMA1 E3 complex. Depletion of JB12 increased CFTR folding efficiency up to threefold and permitted a pool of CFTRΔF508 to fold and escape the ER. Introduction of the V510D misfolding suppressor mutation into CFTRΔF508 modestly increased folding efficiency, whereas combined inactivation of JB12 and suppression of intrinsic folding defects permitted CFTRΔF508 to fold at 50% of wild-type efficiency. Therapeutic correction of CFTRΔF508 misfolding in cystic fibrosis patients may require repair of defective folding kinetics and suppression of ER QC factors, such as JB12.

Ubr1 and Ubr2 function in a quality control pathway for degradation of unfolded cytosolic proteins.

Quality control systems facilitate polypeptide folding and degradation to maintain protein homeostasis. Molecular chaperones promote folding, whereas the ubiquitin/proteasome system mediates degradation. We show here that Saccharomyces cerevisiae Ubr1 and Ubr2 ubiquitin ligases promote degradation of unfolded or misfolded cytosolic polypeptides. Ubr1 also catalyzes ubiquitinylation of denatured but not native luciferase in a purified system. This activity is based on the direct interaction of denatured luciferase with Ubr1, although Hsp70 stimulates polyubiquitinylation of the denatured substrate. We also report that loss of Ubr1 and Ubr2 function suppressed the growth arrest phenotype resulting from chaperone mutation. This correlates with increased protein kinase maturation and indicates partitioning of foldable conformers toward the proteasome. Our findings, based on the efficiency of this quality control system, suggest that the cell trades growth potential to avert the potential toxicity associated with accumulation of unfolded or misfolded proteins. Ubr1 and Ubr2 therefore represent E3 components of a novel quality control pathway for proteins synthesized on cytosolic ribosomes.