ABC-transporter CFTR folds with high fidelity through a modular, stepwise pathway.

The question how proteins fold is especially pointed for large multi-domain, multi-spanning membrane proteins with complex topologies. We have uncovered the sequence of events that encompass proper folding of the ABC transporter CFTR in live cells by combining kinetic radiolabeling with protease-susceptibility assays. We found that CFTR folds in two clearly distinct stages. The first, co-translational, stage involves folding of the 2 transmembrane domains TMD1 and TMD2, plus one nucleotide-binding domain, NBD1. The second stage is a simultaneous, post-translational increase in protease resistance for both TMDs and NBD2, caused by assembly of these domains onto NBD1. Our assays probe every 2-3 residues (on average) in CFTR. This in-depth analysis at amino-acid level allows detailed analysis of domain folding and importantly also the next level: assembly of the domains into native, folded CFTR. Defects and changes brought about by medicines, chaperones, or mutations also are amenable to analysis. We here show that the well-known disease-causing mutation F508del, which established cystic fibrosis as protein-folding disease, caused co-translational misfolding of NBD1 but not TMD1 nor TMD2 in stage 1, leading to absence of stage-2 folding. Corrector drugs rescued stage 2 without rescuing NBD1. Likewise, the DxD motif in NBD1 that was identified to be required for export of CFTR from the ER we found to be required already upstream of export as CFTR mutated in this motif phenocopies F508del CFTR. The highly modular and stepwise folding process of such a large, complex protein explains the relatively high fidelity and correctability of its folding.

A cytosolic reductase pathway is required for efficient N-glycosylation of an STT3B-dependent acceptor site.

N-linked glycosylation of proteins entering the secretory pathway is an essential modification required for protein stability and function. Previously, it has been shown that there is a temporal relationship between protein folding and glycosylation, which influences the occupancy of specific glycosylation sites. Here, we used an in vitro translation system that reproduces the initial stages of secretory protein translocation, folding and glycosylation under defined redox conditions. We found that the efficiency of glycosylation of hemopexin was dependent upon a robust NADPH-dependent cytosolic reductive pathway, which could be mimicked by the addition of a membrane-impermeable reducing agent. We identified a hypoglycosylated acceptor site that is adjacent to a cysteine involved in a short-range disulfide. We show that efficient glycosylation at this site is influenced by the cytosolic reductive pathway acting on both STT3A- and STT3B-dependent glycosylation. Our results provide further insight into the important role of the endoplasmic reticulum redox conditions in glycosylation site occupancy and demonstrate a link between redox conditions in the cytosol and glycosylation efficiency.

The novel HLA-B allele, HLA-B*44:345N, discovered in a Korean family.

The HLA-B*44:345N allele differs from HLA-B*44:03:01:01 by a 19 nucleotide deletion at positions 440 to 458.

Identification of a novel HLA-C*03 variant, C*03:465, using sequence-based typing in a Korean man.

HLA-C*03:465, differs from C*03:04:01:01 by a single nucleotide in codon 135 (GCC → GTC).

A new allele, HLA-B*54:39, identified by sequence-based typing in a Korean individual.

The new allele B*54:39 showed one nucleotide difference from B*54:01:01 in codon 10 (ACC->AGC).

Somatic mutation of HLA-DRB1*04:03 in a patient with myelodysplastic syndrome at diagnosis.

Loss or decrease in expression of human HLA caused by somatic mutations of HLA genes has been reported in various malignancies. However, mutations in the HLA-DR gene have been rarely noted in hematologic malignancies. Here, we report a case of myelodysplastic syndrome (MDS) with a novel point mutation in exon 2 of the HLA-DRB1*04:03 gene pertaining to a silent mutation (c.357A > T[p.Thr=]). When compared before and after anticancer drug treatment and to the results from the full HLA-matching sibling donor, mutation of the HLA-DRB1 gene suggests clonal evolution. In conclusion, we report a new DRB1*04:03 mutation in an MDS patient at diagnosis that results in a synonymous substitution with unknown clinical impact.

Mechanically Robust Magnetic Fe3O4 Nanoparticle/Polyvinylidene Fluoride Composite Nanofiber and Its Application in a Triboelectric Nanogenerator.

Mechanically robust composite nanofibers (NFs) with enhanced magnetic properties were made from polyvinylidene fluoride (PVDF) and Fe3O4 nanoparticles (NPs) by an electrospinning method. At up to 11.3 wt %, Fe3O4 NPs were embedded randomly in the PVDF NFs, but when the content exceeded 17 wt %, the NPs aggregated on the NF surfaces. Magnetization of the composite NFs consistently increased with the increasing Fe3O4 NP content. The mechanical strength of the Fe3O4 NP/PVDF composite NF was enhanced by a dispersion strengthening mechanism. A triboelectric nanogenerator was made from the composite, which showed enhanced output performance with the Fe3O4 NP content less than 11.3 wt %, but the performance degraded at higher content. These results were attributed to the electret doping effect and surface aggregation of the Fe3O4 NPs on the NFs, respectively.

Identification of a warm-temperature acclimation-associated 65-kDa protein encoded by a temperature- and infection-responsive gene in the Kumgang fat minnow Rhynchocypris kumgangensis.

Water temperature is one of the most important factors in fish physiology; thus, it is important to identify genes that respond to changes in water temperature. In this study, we identified a warm- temperature acclimation-associated 65-kDa protein (Wap65) in the Kumgang fat minnow Rhynchocypris kumgangensis, a small, cold-freshwater fish species endemic to Korea. Kumgang fat minnow Wap65-1 (kmWap65-1) was cloned using polymerase chain reaction (PCR)-based strategies, and was found to be highly homologous with teleost Wap65-1 and mammalian hemopexin, a heme-binding protein that transfers plasma heme into hepatocytes. kmWap65-1 mRNA was expressed mainly in the liver and its expression levels were significantly increased by both short- and long-term exposure to high temperature, which was evaluated by real-time quantitative PCR. Furthermore, the expression levels of kmWap65-1 were highly elevated by exposure to bacterial lipopolysaccharide. These results indicate that kmWap65-1 expression is associated with environmental stresses such as increases in water temperature and bacterial infection. J. Exp. Zool. 325A:65-74, 2016. © 2015 Wiley Periodicals, Inc.

Isolation and expansion of synovial CD34(-)CD44(+)CD90(+) mesenchymal stem cells: comparison of an enzymatic method and a direct explant technique.

Synovium-derived mesenchymal stem cells (MSCs) offer a promising therapeutic option for cartilage regeneration. The conventional method of MSC isolation involves single-cell suspensions using collagenases. Recently, a nonenzymatic explant technique was developed to isolate MSCs. We compared these techniques in the isolation of functional MSCs. MSCs were isolated from human fibrous and adipose synovium of osteoarthritic patients using explants or enzymatic methods. Total cell number, percentage of MSCs, and surface marker expression of MSCs were measured following expansion. Multipotentiality was determined using a MSC functional identification kit. MSCs isolated from fibrous or adipose synovium using these two techniques expressed similar levels of the surface markers CD44, CD90, and CD105, and displayed similar multipotentiality in generating adipocytes, osteoblasts, and chondrocytes. Total cell number and number of CD34(-)CD44(+)CD90(+) MSCs after 10-day expansion were similar in each culture, regardless of the source and method used, although the percentage of MSCs was slightly higher in explant cultures. There were no correlations between MSC yield and patient age, Hospital for Special Surgery score, and degree of deformity under all culture conditions. Both the enzymatic and explant techniques yielded similar yields of MSCs with similar characteristics. Because the explant technique is simpler and less invasive, it may be preferred over enzymatic techniques for isolating MSCs from the synovium of osteoarthritic patients for cartilage regeneration.