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Divergent signaling via SUMO modification: potential for CFTR modulation. Am J Physiol Cell Physiol 310: C175-C180, 2016; doi:10.1152/ajpcell.00124.2015.--The cystic fibrosis transmembrane conductance regulator (CFTR) is generally responsible for the cAMP/PKA regulated anion conductance at the apical membranes of secretory epithelial cells. Mutations in CFTR underlie cystic fibrosis (CF), in which the most common variant, F508del, causes protein misfolding and its proteasome-mediated degradation. A new pathway that contributes to mutant CFTR degradation is mediated by the small heat shock protein, Hsp27, which cooperates with Ubc9, the E2 enzyme for SUMOylation, to selectively conjugate mutant CFTR with SUMO-2/3. This SUMO paralog can form polychains, which are recognized by the ubiquitin E3 enzyme, RNF4, leading to CFTR ubiquitylation and recognition by the proteasome. We found also that F508del CFTR could be modified by SUMO-1, a paralog that does not support SUMO polychain formation. The use of different SUMO paralogs to modify and target a single substrate for divergent purposes is not uncommon. In this short review we discuss the possibility that conjugation with SUMO-1 could protect mutant CFTR from disposal by RNF4 and similar ubiquitin ligases. We hypothesize that such a pathway could contribute to therapeutic efforts to stabilize immature mutant CFTR and thereby enhance the action of therapeutics that correct CFTR trafficking to the apical membranes. [ABSTRACT FROM AUTHOR]

A newly identified pathway for selective degradation of the common mutant of the cystic fibrosis transmembrane conductance regulator (CFTR), F508del, is initiated by binding of the small heat shock protein, Hsp27. Hsp27 collaborates with Ubc9, the E2 enzyme for protein SUMOylation, to selectively degrade F508del CFTR via the SUMO-targeted ubiquitin E3 ligase, RNF4 (RING finger protein 4) (1). Here, we ask what properties of CFTR are sensed by the Hsp27-Ubc9 pathway by examining the ability ofNBD1(locus of the F508del mutation) to mimic the disposal of full-length (FL) CFTR. Similar to FL CFTR, F508del NBD1 expression was reduced 50-60% by Hsp27; it interacted preferentially with the mutant and was modified primarily by SUMO-2. Mutation of the consensus SUMOylation site, Lys447, obviated Hsp27-mediated F508del NBD1 SUMOylation and degradation. As for FL CFTR and NBD1 in vivo, SUMO modification using purified components in vitro was greater for F508del NBD1 versusWTand for the SUMO-2 paralog. Several findings indicated that Hsp27-Ubc9 targets the SUMOylation of a transitional, non-native conformation of F508del NBD1: (a) its modification decreased as [ATP] increased, reflecting stabilization of the nucleotide-binding domain by ligand binding; (b) a temperature-induced increase in intrinsic fluorescence, which reflects formation of a transitionalNBD1conformation, was followed by its SUMO modification; and (c) introduction of solubilizing or revertant mutations to stabilize F508del NBD1 reduced its SUMO modification. These findings indicate that the Hsp27-Ubc9 pathway recognizes a non-native conformation of mutant NBD1, which leads to its SUMO-2 conjugation and degradation by the ubiquitin-proteasome system. [ABSTRACT FROM AUTHOR]

Alpine regions’ groundwater is crucial to the worldwide hydrological cycle. However, due to the harsh environmental conditions, the distribution and evolution characteristics await clarification. The study area was selected to be the Nagqu River Basin in the Nu-Salween River’s source region. In 2019-2021, we gathered 88,000 monitoring data from nine observation wells and examined the spatiotemporal groundwater table changes in various permafrost zones and freeze-thaw cycles. During the freezing period, entirely frozen period, thawing period, and entirely thawed period, the groundwater table change rates in the permafrost zone were 2.14, 1.54, 1.55, and 2.01 times larger than in the seasonal frost zone, and fluctuation amplitudes were 1.97, 1.28, 1.01 and 1.31 times larger. The average groundwater table change rate and fluctuation amplitude were greatest during the entirely thawed period and lowest during the thawing period, with the maximum change rate reaching 3.64 cm/d during the entirely thawed period of 2019-2020 in the permafrost zone and the minimum change rate of 0.12 cm/d during the thawing period of 2019-2020 in the seasonal frost zone. [ABSTRACT FROM AUTHOR]

The elevated expression of urokinase-type plasminogen activator receptor (uPAR) is associated with the poor prognosis of pancreatic cancer patients. Thus, uPAR is a promising candidate as a molecular target for the non-invasive imaging of pancreatic cancer. The present study aimed to develop a technetium-99m (99mTc)-labeled uPAR-binding peptide for non-invasive single-photon emission computed tomography (SPECT) assessment of uPAR expression in pancreatic cancer xenograft models. A linear high-affinity uPAR peptide antagonist, Hynic-PEG-AE105, was labeled with 99mTc. Human uPAR-positive pancreatic cancer BxPC-3 cells were inoculated into nude mice. SPECT was performed in the pancreatic cancer xenograft mice models. The results showed that the rate of the 99mTc labeling of Hynic-PEG-AE105 was 97.72±1.73%. The tumor uptake of 99mTc-Hynic-PEG-AE105 was higher than the control inactive peptide 99mTc-Hynic-PEG-AE105mut at 4 h (3.37±0.11 vs. 1.36±0.18; P<0.001) and 6 h (3.64±0.25 vs. 1.28±0.20; P<0.001) (n=10). Moreover, a significant correlation was observed between the tumor uptake of 99mTc-Hynic-PEG-AE105 and uPAR expression (r=0.791, P=0.006). In conclusion, in the present study, a peptide-based SPECT tracer, 99mTc-Hynic-PEG-AE105, with a high purity and specific radioactivity was synthesized. 99mTc-Hynic-PEG-AE105 is a promising agent for the non-invasive determination of uPAR expression in pancreatic cancer. [ABSTRACT FROM AUTHOR]

Generating an artificial blood flow in a circle system is an important step in hemodynamic research; thus, the influence of circle system components on the pulsatile flow wave forms should be investigated. In this study, a circle system was built, in which two solenoid valves were controlled by a timer to transform a constant flow into a pulsatile one, and two customized compliances with five different aeration volumes were set up upstream and downstream of the test chamber, achieving twenty-five different wave forms. Then, the influence of the compliance settings on the flow rate wave form was investigated. From the experimental data, it can be concluded that the absolute value of the maximum value and the minimum value of the wave forms increases along the aeration of the downstream compliance but decreases along the aeration of the upstream compliance. For the second maximum value and the offset between the maximum value and the minimum value, remarkable differences were obtained between runs with aeration of zero compliance and nonzero compliance. Finally, an emulational flow was achieved with the up- and downstream aeration volume equaling 360mL and 180mL, respectively, which fit the realistic wave form well. [ABSTRACT FROM AUTHOR]

The cell response to virus infection and virus perturbation of that response is dynamic and is reflected by changes in cell susceptibility to infection. In this study, we evaluated the response of human epithelial cells to sequential infections with human respiratory syncytial virus strains A2 and B to determine if a primary infection with one strain will impact the ability of cells to be infected with the second as a function of virus strain and time elapsed between the two exposures. Infected cells were visualized with fluorescent markers, and location of all cells in the tissue culture well were identified using imaging software. We employed tools from spatial statistics to investigate the likelihood of a cell being infected given its proximity to a cell infected with either the homologous or heterologous virus. We used point processes, K-functions, and simulation procedures designed to account for specific features of our data when assessing spatial associations. Our results suggest that intrinsic cell properties increase susceptibility of cells to infection, more so for RSV-B than for RSV-A. Further, we provide evidence that the primary infection can decrease susceptibility of cells to the heterologous challenge virus but only at the 16 h time point evaluated in this study. Our research effort highlights the merits of integrating empirical and statistical approaches to gain greater insight on in vitro dynamics of virus-host interactions. [ABSTRACT FROM AUTHOR]

Mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene cause cystic fibrosis (CF). The most common mutation, AF508, omits the phenylalanine residue at position 508 in the first nucleotide binding domain (NBD1) of CFTR. The mutant protein is retained in the endoplasmic reticulum and degraded by the ubiquitin-proteasome system. We demonstrate that expression of NBD1 plus the regulatory domain (RD) of ΔF508 CFTR (ΔFRD) restores the biogenesis of mature ΔF508 CFTR protein. In addition, AFRD elicited a cAMP-stimulated anion conductance response in primary human bronchial epithelial (HBE) cells isolated from homozygous ΔF508 CF patients. A protein transduction domain (PTD) could efficiently transduce (~90%) airway epithelial cells. When fused to a PTD, direct addition of the AFRD peptide conferred a dose-dependent, cAMP-stimulated anion efflux to ΔF508 HBE cells. Hsp70 and Hsp90 associated equally with WT and ΔF508 CFrR, whereas nearly twice as much of the Hsp90 cochaperone, Ahal, associated with AF508 CFTR. Expression of AFRD produced a dose-dependent removal of Ahal from AF508 CFTR that correlated with its functional rescue. These findings indicate that disruption of the excessive association of the cochaperone, Ahal, with AF508 CFTR is associated with the correction of its maturation, trafficking and regulated anion channel activity in human airway epithelial cells. Thus, PTD-mediated ΔFRD fragment delivery may provide a therapy for CF. [ABSTRACT FROM AUTHOR]

BACKGROUND: The objective was to determine risk factors of hepatitis C virus (HCV) infection in blood donors in Beijing. STUDY DESIGN AND METHODS: A cross-sectional study was conducted in a total of 112 volunteer blood donors at Beijing Red Cross Blood Center (BRCBC) who were identified between January 2002 and December 2003 due to isolated HCV antibody reactivity at a donor screening procedure. The donors were recalled and interviewed about possible risk factor history. Additional blood samples were tested in repeated enzyme immunoassay, recombinant immunoblot assay, and nested reverse transcription–polymerase chain reaction. Chi-square test, analysis of variance, and multivariate logistic regression analysis were conducted to examine associations between HCV serostatus and potential risk factors. RESULTS: Among donors identified with isolated HCV antibody reactivity at donor screening, the prevalence of confirmed HCV infection was 31.3 percent (95% confidence interval [CI], 22.0%-39.2%) and indeterminate HCV infection was 22.3 percent (95% CI, 14.7%-30.3%), and the negative prevalence was 46.4 percent (95% CI, 37.5%-56.1%). The confirmed HCV cases were more likely than the indeterminate HCV cases to have the histories of previous transfusion, plasma donation, and hepatitis infection or elevated alanine aminotransferase (ALT), whereas the HCV-negative subjects were least likely to have the above histories (p < 0.05). CONCLUSION: Histories of blood transfusion before 1995, plasma donation, previous hepatitis infection, or elevated ALT were risk factors for HCV infection among blood donors in Beijing. [ABSTRACT FROM AUTHOR]

Background and Objectives: The impacts of nutritional status on clinical outcomes in children receiving umbilical cord blood stem cell transplantation (UCBT) are not fully described. We evaluated the risk for malnutrition before transplantation admission and influence of weight loss during hospitalization on short-term clinical outcomes in children with UCBT. Methods and Study Design: We conducted a retrospective study of pediatric patients up to age 18 years who received UCBT and were treated at the Children’s Hospital of Fudan University between January 2019 and December 2020. Results: The mean age of the 91 patients was 1.3 years, with 78 (85.7%) men and 13 (14.3%) women (p<0.001). UCBT was performed mostly for primary immunodeficiency disease (PID) (83, 91.2%). The weight loss differences among children with different primary diseases were statistically significant (p=0.003). Children with a large amount of weight loss during hospitalization (n = 24) had higher risks of skin graft-versus-host disease (GVHD) (multivariate OR=5.01, 95% CI: 1.35-18.65), intestinal GVHD (multivariate OR=7.27, 95% CI: 1.74-30.45), a longer median hospital stay (p=0.004), higher antibiotic costs (p=0.008) and higher total hospitalization costs (p=0.004). Malnutrition on admission was significantly positively correlated with longer parenteral nutrition (PN) time (p=0.008). Early nutritional intervention effects on clinical outcomes need further assessment. Conclusions: Underweight recipient child and excessive weight loss during transplantation increases the length and cost of hospital stay, and is associated with a high incidence of GVHD, which affects the prognosis of transplantation and medical resources consumption. [ABSTRACT FROM AUTHOR]

A complex involving Derlin-1 and p97 mediates the retro-translocation and endoplasmic reticulum (ER)-associated degradation of misfolded proteins in yeast and is used by certain viruses to promote host cell protein degradation (Romisch, K. (2005) Annu. Rev. Cell Dev. Biol. 21, 435- 456; Lilley, B. N., and Ploegh, H. L. (2004) Nature 429, 834-840; Ye, Y., Shibata, Y., Yun, C., Ron, D., and Rapoport, T. A. (2004) Nature 429, 841-847). We asked whether the components of this pathway are involved in the endoplasmic reticulum-associated degradation of the mammalian integral membrane protein, the cystic fibrosis transmembrane conductance regulator (CFTR), a substrate for the ubiquitin-proteasome system. We report that Derlin-1 and p97 formed complexes with CFTR in human airway epithelial cells. Derlin-1 interacted with nonubiquitylated CFTR, whereas p97 associated with ubiquitylated CFTR. Exogenous expression of Derlin-1 led to its co-localization with CFTR in the ER where it reduced wild type (WT) CFTR expression and efficiently degraded the disease-associated CFTR folding mutants, ΔF508 and G85E (>90%). Consistent with this, Derlin-1 also reduced the amount of WT or ΔF508 CFTR appearing in detergent-insoluble aggregates. An ~70% knockdown of endogenous Derlin-1 by RNA interference increased the steady-state levels of WT and ΔF508 CFTR by 10-15-fold, reflecting its significant role in CFTR degradation. Derlin-1 mediated the degradation of N-terminal CFTR fragments corresponding to the first trans-membrane domain of CFTR, but CFTR fragments that incorporated additional domains were degraded less efficiently. These findings suggest that Derlin-1 recognizes misfolded, nonubiquitylated CFTR to initiate its dislocation and degradation early in the course of CFTR biogenesis, perhaps by detecting structural instability within the first transmembrane domain. [ABSTRACT FROM AUTHOR]

Unesterified cholesterol accumulates in late endosomes in cells expressing the misfolded cystic fibrosis transmembrane conductance regulator (CFTR). CFTR misfolding in the endoplasmic reticulum (ER) or general activation of ER stress led to dynein-mediated clustering of cholesterol-loaded late endosomes at the Golgi region, a process regulated by ER-localized VAMP-associated proteins (VAPs).Wehypothesized that VAPs serve as intracellular receptors that couple lipid homeostasis through interactions with two phenylalanines in an acidic track (FFAT) binding signals (found in lipid sorting and sensing proteins, LSS) with proteostasis regulation. VAPB inhibited the degradation of ΔF508-CFTR. The activity was mapped to the ligand-binding major sperm protein (MSP) domain, which was sufficient in regulating CFTR biogenesis. We identified mutations in an unstructured loop within the MSP that uncoupled VAPB-regulated CFTR biogenesis from basic interactions with FFAT. Using this information, we defined functional and physical interactions between VAPB and proteostasis regulators (ligands), including the unfolded protein response sensor ATF6 and the ER degradation cluster that included FAF1, VCP, BAP31, and Derlin-1. VAPB inhibited the degradation of ΔF508-CFTR in the ER through interactions with the RMA1- Derlin-BAP31-VCP pathway. Analysis of pseudoligands containing tandem FFAT signals supports a competitive model for VAP interactions that direct CFTR biogenesis. The results suggest a model in which VAP-ligand binding couples proteostasis and lipid homeostasis leading to observed phenotypes of lipid abnormalities in protein folding diseases. [ABSTRACT FROM AUTHOR]