The Relationship of Energy Malnutrition, Skeletal Muscle and Physical Functional Performance in Patients with Stable Chronic Obstructive Pulmonary Disease

Weight loss is a factor that affects prognosis in patients with chronic obstructive pulmonary disease (COPD) independent of lung function. One of the major factors for weight loss is energy malnutrition. There have been no reports on the factors related to energy malnutrition in COPD patients. This retrospective observational study aimed to investigate these factors. We included 163 male subjects with COPD. Respiratory quotient (RQ), an index of energy malnutrition, was calculated by expiratory gas analysis using an indirect calorimeter. RQ 2). Energy malnutrition was observed in 43%. The independent factors associated with energy malnutrition were tidal volume (VT) (OR 0.99; 95% CI 0.985–0.998; p = 0.015) and Th12 erector spinae muscle cross-sectional area SMI (Th12ESMSMI) (OR 0.71; 95% CI 0.535–0.946; p = 0.019). In decision-tree profiling of energy malnutrition, VT was extracted as the first distinguishable factor, and Th12ESMSMI as the second. In ROC analysis, VT SMI < 10.1 (AUC, 0.70) was the cutoff value for energy malnutrition. Energy malnutrition may be an early warning sign of nutritional disorders

Sec16A, a key protein in COPII vesicle formation, regulates the stability and localization of the novel ubiquitin ligase RNF183

<div><p>We identified 37 ubiquitin ligases containing RING-finger and transmembrane domains. Of these, we found that RNF183 is abundantly expressed in the kidney. RNF183 predominantly localizes to the endoplasmic reticulum (ER), Golgi, and lysosome. We identified Sec16A, which is involved in coat protein complex II vesicle formation, as an RNF183-interacting protein. RNF183 colocalized with Sec16A and interacted through the central conserved domain (CCD) of Sec16A. Although Sec16A is not a substrate for RNF183, RNF183 was more rapidly degraded by the ER-associated degradation (ERAD) in the absence of Sec16A. Sec16A also stabilized the interacting ubiquitin ligase RNF152, which localizes to the lysosome and has structural similarity with RNF183. These results suggest that Sec16A appears to regulate the protein stability and localization of lysosomal ubiquitin ligases.</p></div

Proteomics analysis of the RNF183 interacting protein.

<p>Proteomics analysis of the RNF183 interacting protein.</p

Interaction and colocalization of RNF183 and Sec16A.

<p>(A) Interaction of RNF183 and Sec16A. Cell lysates from HEK293 cells stably expressing V5-tagged RNF183 were immunoprecipitated using anti-V5 antibody, and the immune complexes were analyzed by Western blotting with anti-Sec16A (<i>upper</i> panel) or anti-V5 (<i>lower</i> panel) antibodies. (B) Colocalization of RNF183 with Sec16A. HeLa cells stably transfected with RNF183-V5 were subjected to immunofluorescence staining with anti-Sec16A (<i>green</i>) and DAPI (<i>blue</i>). (C) Effect of RNF183 knockdown on Sec16A protein. RNF183 expression in HEK293 cells stably transfected with RNF183-V5 was suppressed using siRNA. Endogenous Sec16A protein levels were detected using Western blotting with anti-Sec16A antibody. NC, negative control. (D) Schematic diagram of the predicted domains of Sec16A. (E) Interaction domain of Sec16A with RNF183. Lysates from HEK293 cells stably expressing RNF183 transiently transfected with GFP-tagged full-length Sec16A (Full) or its deletion mutant constructs were subjected to immunoprecipitation with anti-V5 antibody, followed by immunoblotting with anti-GFP antibody.</p

Effects of Sec16 on other ubiquitin ligases.

<p>(A) Interactions of RNF152 and HRD1 with Sec16A. Coimmunoprecipitation was performed in HEK293 cells engineered to stably express V5-tagged RNF183, V5-tagged RNF152, or myc-tagged HRD1. Cell lysates were immunoprecipitated with anti-V5 or anti-myc antibodies or normal mouse immunoglobulin G (IgG; negative control). Immune complexes were analyzed by Western blotting with an anti-Sec16A antibody (<i>top</i> panel) and anti-V5 (<i>second</i> panel) or anti-myc antibodies (<i>third</i> panel). (B, D) Effect of Sec16A downregulation on RNF152 and HRD1 protein stability. Stable RNF152-V5- or HRD1-myc-expressing HEK293 cells were transfected with NC or Sec16A siRNA. At 48 h after transfection, cells were subjected to a CHX assay. (C, E) Asterisks represent significant differences (n = 3; Student’s t test with Bonferroni correction, *p < 0.05, ***p < 0.001; NC vs. Sec16A siRNA).</p

Effects of Sec16 on RNF183 subcellular localization.

<p>(A) Effect of Sec16A downregulation on RNF183 subcellular localization. HeLa cells stably expressing RNF183-V5 were transfected with NC (1st, 3rd, 5th panels) or Sec16A (2nd, 4th, 6th panels) siRNAs. At 48 h after transfection, cells were subjected to immunofluorescence staining with anti-V5 (<i>green</i>) and anti-calnexin, GM130, or LAMP1 (<i>red</i>) antibodies, and DAPI (<i>blue</i>). (B) Effect of proteasome inhibition on RNF183 subcellular localization. HeLa cells stably expressing RNF183-V5 were transfected with NC (<i>top</i> panels) or Sec16A (<i>middle</i> and <i>bottom</i> panels) siRNAs. At 36 h after transfection, cells were incubated with (<i>bottom</i> panels) or without (<i>top</i> and <i>middle</i> panels) 10 μM MG132 for 12 h.</p

Effects of Sec16 on RNF183 protein stability.

<p>(A) Effect of Sec16A downregulation on RNF183 protein stability. HeLa cells stably expressing RNF183-V5 were transfected with NC (1st, 3rd, and 5th panels) or Sec16A (2nd, 4th and 6th panels) siRNA. At 44 h after transfection, cells were treated with 30 μg/ml cycloheximide (CHX) and 10 μM MG132 for the indicated periods. Total cell lysates were analyzed by Western blotting with an anti-V5 (1st and 2nd panels), Sec16A (3rd and 4th panels), and β-actin (5th and 6th panels) antibodies. (B) Quantitative curves of data from (A). RNF183 levels at each time point were plotted relative to the level at time 0 (n = 3). Asterisks represent significant differences (Student’s t test with Bonferroni correction, *p < 0.05; NC vs. Sec16A siRNA; #p < 0.05, ##p < 0.01; ##p < 0.001; Sec16A siRNA vs. Sec16A siRNA + MG132).</p