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Autophagosome formation is the hallmark of macroautophagy (herein referred to as autophagy) and requires the covalent conjugation of LC3 proteins (or Atg8 in yeast) to the amino headgroup of PE (phosphatidylethanolamine) lipids. Atg3 is an enzyme that catalyzes the final step of this reaction by transferring LC3 from an LC3-Atg3 intermediate to PEs in targeted membranes. Here, we determine the solution structure of human Atg3 (hAtg3) and demonstrate that the catalytically important regions of hAtg3 are conformationally dynamic. Furthermore, we reveal that these regions and hAtg3’s N-terminal membrane curvature-sensing amphipathic helix concurrently interact with the membrane. These structural studies indicate that hAtg3 exploits a multifaceted membrane-association mechanism to position its catalytic center at the membrane surface and to bring the reaction substrates of LC3 and PE lipids to proximity for effective LC3-PE conjugation. In addition, our studies demonstrate that the interaction of the His266 residue with the membrane is primarily responsible for hAtg3’s pH-dependent activity. Our investigations advance an emerging concept that the interactions of Atg3 with the highly curved membrane rims of the phagophore spatially regulate autophagosome biogenesis.

VPS37A, a component of ESCRT-I, is essential for recruiting a subset of ESCRT proteins that seal the phagophore during autophagosome biogenesis. In this study, we uncover two hydrophobic motifs in the VPS37A N-terminal 148 amino acids (VPS37A1-148) that selectively interact with highly curved membranes. Mutations in these motifs nearly abolish VPS37A membrane bindingin vitroand compromise its localization to the phagophore and autophagic fluxin vivo. We also determined the solution structure of residues 21 to 131 and demonstrated that it is the UEVL (ubiquitin E2 variant-like) domain. Intriguingly, this domain remodels highly curved liposomes to high-order structures. We suggest that the specific interactions between VPS37A1-148and the curved membrane may facilitate the recruitment of VPS37A to the phagophore and its subsequent closure. Our results support the premise that the distinct membrane architecture of the cup-like phagophore spatiotemporally regulates autophagosome biogenesis.

Background General self-efficacy is considered one of the most influential parameters affecting the quality of clinical practice and nurses’ perceived professional benefits (NPPB). Perceived organizational support (POS) is regarded as being central in understanding job-related attitudes, and it is important to enhance POS for nurses to maintain their current employment. NPPB can further reduce nurses’ job burnout and turn-over intention. Many studies have explored the relationships among general self-efficacy, POS, nursing practice environment (NPE) and NPPB. However, a moderating effect of NPE has not been fully explored in nurses, especially among paediatric nurses. Methods A descriptive cross-sectional study was conducted from July to October 2018 with 300 paediatric nurses from 3 Class A tertiary hospitals in Jilin Province. The respondents completed the General Self-Efficacy Scale, Perceived Organizational Support Scale, Practice Environment Scale and Nurses’ Perceived Professional Benefits Scale. The data were analyzed using path analysis and SPSS (version 23.0, IBM). Results General self-efficacy and POS were significantly positively associated with NPPB, which showed that the model had a good fit to the data. NPE was found to play a partial mediating role between POS and NPPB and also had a complete mediating role between general self-efficacy and NPPB. Conclusions The results suggest that general self-efficacy indirectly influences NPPB, and POS directly and indirectly influences NPPB by NPE. Effective measures should be taken to improve nurses’ practice environment in hospitals to raise nurses’ enthusiasm and confidence in their work.

Selective autophagy sequesters cytoplasmic cargo for lysosomal degradation via the binding of autophagy receptors to Atg8 (autophagy-related 8) family proteins on the autophagic membrane. The sole yeast Atg8 gene has six mAtg8 (mammalian Atg8) homologs, including the MAP1LC3 (microtubule-associated protein-1 light chain 3) family and the GABA receptor–associated proteins. Selective autophagy receptors interact with two conserved hydrophobic pockets (termed the W-site and L-site) of mATG8 proteins through a linear motif called the LC3-interacting region (LIR) with the general composition (W/F/Y)XX(I/L/V). To address a lack in our knowledge regarding LIR peptide specificity toward each mATG8 homolog, here we used competitive time-resolved FRET to sensitively and quantitatively characterize the interactions between LIRs and mAtg8. We report that 14 representative LIR-containing peptides display differential binding affinities toward the mAtg8 proteins and identified the LIR domain peptide of TP53INP1 as exhibiting high affinity for all six mATG8 proteins. Using peptide truncation studies, we found that both N- and C-terminal acidic residues, as well as the C-terminal Cys residue of the TP53INP1 LIR peptide, are required for its high-affinity binding to LC3A and LC3B, whereas binding to the GABARAP subfamily proteins was facilitated by residues either N-terminal or C-terminal to the core motif. Finally, we used NMR chemical shift perturbation analysis to gain molecular insights into these findings. Collectively, our results may aid in the development of molecules that selectively disrupt specific mATG8–LIR interactions to dissect the biological roles of the six mATG8 homologs for potential therapeutic applications.

Proteins are often tagged for visualization or delivery in the “sea” of other macromolecules in cells but how tags affect protein mobility remains poorly understood. Here, we employ in-cell 19F NMR...

Human Atg3 (hAtg3) is an E2-like enzyme that catalyzes the conjugation of LC3 family proteins to phosphatidylethanolamine (PE) lipids in the autophagosomal membrane during autophagy. The reaction product, LC3-PE, acts as a marker for autophagic cargo and is required for the effective construction of functional autophagosomes. However, the structural and molecular basis of this conjugation reaction remains elusive, at least in part, because of the absence of lipid bilayers in structural studies conducted to date. Here, we report a sequential resonance assignment for an hAtg3 construct both in aqueous solution and in bicelles. hAtg3 has 314 residues, and our construct lacks the unstructured region from residues 90 to 190. Our results demonstrate a structural rearrangement of hAtg3 N-terminus when it interacts with membranes.

Context Health-related quality of life (QOL) is a recommended clinical tool to assess hemodialysis patients and a primary end point to observe the effectiveness of overall disease management. Empathy is associated with positive outcomes such as pain relief and reduced anxiety and distress. Numerous studies have tested the relationships among empathy, forgiveness, and QOL; however, a mechanism of forgiveness has not been fully explored in hemodialysis patients. Objectives To test the relationship among empathy and health-related QOL and confirm the moderating effects of forgiveness on relationship between empathy and health-related QOL among hemodialysis patients. Methods In a descriptive cross-sectional study conducted from September to December 2017, 457 hemodialysis patients from five hospitals filled out the Heartland Forgiveness Scale, Interpersonal Reactivity Index-C, Kidney Disease Questionnaire, and general information. The data were analyzed using SPSS, and structural equation modeling was used to address the relationships among empathy, forgiveness, and health-related QOL. Results Empathy was significantly positively associated with health-related QOL. The proposed model had a good fit to the data. Forgiveness was found to play a partial mediating role between empathy and health-related QOL. Conclusion The results imply that empathy significantly directly and indirectly influences health-related QOL. Empathy among hemodialysis patients should be monitored and effectively managed to improve positive effects on their health-related QOL. Nurses should consider implementing empathy interventions with an emphasis on building forgiveness strategies to help hemodialysis patients improve their health-related QOL.

Protein diffusion in living cells might differ significantly from that measured in vitro. Little is known about the effect of globular protein size on rotational diffusion in cells because each protein has distinct surface properties, which result in different interactions with cellular components. To overcome this problem, the B1 domain of protein G (GB1) and several concatemers of the protein were labeled with 5-fluorotryptophan and studied by 19F NMR in Escherichia coli cells, Xenopus laevis oocytes, and in aqueous solutions crowded with glycerol, or Ficoll70™ and lysozyme. Relaxation data show that the size dependence of protein rotation in cells is due to weak interactions of the target protein with cellular components, but the effect of these interactions decreases as protein size increases. The results provide valuable information for interpreting protein diffusion data acquired in living cells. Graphical abstract Size matters. The protein rotational mobility in living cells was assessed by 19F NMR. The size dependence effect may arise from weak interactions between protein and cytoplasmic components.

Proteins are often tagged for visualization or delivery in the "sea" of other macromolecules in cells but how tags affect protein mobility remains poorly understood. Here, we employ in-cell

During the last two decades NMR methods have been rapidly developed to study proteins in living Escherichia coli cells. This chapter reviews the labeling strategies and sample preparations for protein NMR in E. coli and summarizes some of the key advances including the determination of protein high-resolution 3D structures and the characterization of protein interactions and stabilities in E. coli cells. In addition, the limitations and future applications are discussed.

During the last decade 19F NMR has been developed as a popular tool for biomolecular NMR in living cells. Because there is no background perturbation, high sensitivity or large chemical shift dispersion, this spin-1/2 19F nucleus has been selectively incorporated into macrobiomolecules such as proteins and nucleic acids to study their conformation, interactions, and dynamics in a complex cellular environment. This chapter reviews 19F labeling methods for in-cell NMR and summarizes some of their applications in proteins and nucleic acid studies in cells.

Clinical performance is regarded as a basic and core content of nursing quality. Professional values of nursing students may play an important role in improving clinical performance. Few studies reveal the relationship between professional values, perceived stress and clinical performance. This research has found that professional values play a moderating role in the relationship between perceived stress and clinical performance.To examine the negative association between perceived stress and clinical performance among practice nursing students, and to determine whether professional values plays a moderating role in the relationship between perceived stress and clinical performance among Chinese practice nursing students.A cross-sectional descriptive design has been used.From May to June 2017, a total of 435 Chinese practice nursing students from 4 hospitals filled out the questionnaires. The response rate was 94.57%. Three questionnaires including the Perceived Stress Scale (PSS), Nursing Professional Value Scale-Revised (NPVS-R), and Clinical Performance Scale (CPS). Structural equation modeling was used to deal with the relationships among perceived stress, professional values, and clinical performance.Perceived stress was negatively associated with clinical performance (P 0.01). Professional values significantly affected clinical performance (P 0.01) and moderated the relationship between perceived stress and clinical performance (P 0.01).Perceived stress is negatively related to clinical performance among Chinese practice nursing students, and professional values moderates the relationship between perceived stress and clinical performance, which can form scientific evidence to develop intervention strategies to improve clinical performance.

Protein diffusion in living cells might differ significantly from that measured in vitro. Little is known about the effect of globular protein size on rotational diffusion in cells because each protein has distinct surface properties, which result in different interactions with cellular components. To overcome this problem, the B1 domain of protein G (GB1) and several concatemers of the protein were labeled with 5-fluorotryptophan and studied by

Investigating the structure, modification, interaction, and function of biomolecules in their native cellular environment leads to physiologically relevant knowledge about their mechanisms, which will benefit drug discovery and design. In recent years, nuclear and electron magnetic resonance (NMR) spectroscopy has emerged as a useful tool for elucidating the structure and function of biomacromolecules, including proteins, nucleic acids, and carbohydrates in living cells at atomic resolution. In this review, we summarize the progress and future of in-cell NMR as it is applied to proteins, nucleic acids, and carbohydrates.

The Ca(2+) -mediated conformational transition of the protein calmodulin (CaM) is essential to a variety of signal transduction pathways. Whether the transition in living cells is similar to that observed in buffer is not known. Here, we report the direct observation by (19) F NMR spectroscopy of the transition of the Ca(2+) -free and -bound forms in Xenopus laevis oocytes at different Ca(2+) levels. We find that the Ca(2+) -bound CaM population increased greatly upon binding the target protein myosin light-chain kinase (MLCK) at the same Ca(2+) level. Paramagnetic NMR spectroscopy was also exploited for the first time to obtain long-range structural constraints in cells. Our study shows that (19) F NMR spectroscopy can be used to obtain long-range structural constraints in living eukaryotic cells and paves the way for quantification of protein binding constants.

The X-chromosome linked inhibitor of apoptosis (XIAP) is a multidomain metalloprotein involved in caspase inhibition and in copper homeostasis. It contains three zinc-binding baculoviral IAP repeats (BIR) domains, which are responsible for caspase interaction. Recently, it has been suggested that the BIR domains can bind copper, however high resolution data on such interaction is missing. Here we characterize by NMR the structural properties of BIR1 in solution, and the effects of its interaction with copper both in vitro and in physiological environments. BIR1 is dimeric in solution, consistent with the X-ray structure. Cysteine 12, located in the unfolded N-terminal region, has a remarkably low redox potential, and is prone to oxidation even in reducing physiological environments. Interaction of BIR1 with copper(II) results in the oxidation of cysteine 12, with the formation of either an intermolecular disulfide bond between two BIR1 molecules or a mixed disulfide bond with glutathione, whereas the zinc binding site is not affected by the interaction.

In-cell NMR spectroscopy provides insight into protein conformation, dynamics, and function at atomic resolution in living cells. Systematic evaluation of isotopic-labeling strategies is necessary to observe the target protein in the sea of other molecules in the cell. Here, we investigate the detectability, sensitivity, and resolution of in-cell NMR spectra of the globular proteins GB1, ubiquitin, calmodulin, and bcl-xl-cutloop, resulting from uniform (15)N enrichment (with and without deuteration), selective (15)N-Leu enrichment, (13)C-methyl enrichment of isoleucine, leucine, valine, and alanine, fractional (13)C enrichment, and (19)F labeling. Most of the target proteins can be observed by (19)F labeling and (13)C enrichment with direct detection because selectively labeling suppresses background signals and because deuteration improves in-cell spectra. Our results demonstrate that the detectability of proteins is determined by weak interactions with intercellular components and that choosing appropriate labeling strategies is critical for the success of in-cell protein NMR studies.

Protein mobility in living cells is vital for cell function. Both cytosolic viscosity and weak protein-protein interactions affect mobility, but examining viscosity and weak interaction effects is challenging. Herein, we demonstrate the use of (19) F NMR spectroscopy to measure cytoplasmic viscosity and to characterize nonspecific protein-protein interactions in living Escherichia coli cells. The origins of resonance broadening in Escherichia coli cells were also investigated. We found that sample inhomogeneity has a negligible effect on resonance broadening, the cytoplasmic viscosity is only about 2-3 times that of water, and ubiquitous transient weak protein-protein interactions in the cytosol play a significant role in governing the detection of proteins by using in-cell NMR spectroscopy.