Distinct Mechanisms for Processing Autophagy Protein LC3-PE by RavZ and ATG4B

Autophagy is a conserved catabolic process involved in the elimination of proteins, organelles and pathogens in eukaryotic cells. Lipidated LC3 proteins that are conjugated to phosphatidylethanolamine (PE) play a key role in autophagosome biogenesis. Endogenous ATG4-mediated deconjugation of LC3-PE is required for LC3 recycling. However, the Legionella effector RavZ irreversibly deconjugates LC3-PE to inhibit autophagy. It is not clear how ATG4 and RavZ process LC3-PE with distinct modes. Herein, a series of semisynthetic LC3-PE proteins containing C-terminal mutations or insertions were used to investigate the relationship of the C-terminal structure of LC3-PE with ATG4/RavZ-mediated deconjugation. Using a combination of molecular docking and biochemical assays, we found that Gln116, Phe119 and Gly120 of LC3-PE are required for cleavage by both RavZ and ATG4B, whereas Glu117(LC3) is specific to cleavage by RavZ. The molecular ruler mechanism exists in the active site of ATG4B, but not in RavZ. Met63 and Gln64 at the active site of RavZ are involved in accommodating LC3 C-terminal motif. Our findings show that the distinct binding modes of the LC3 C-terminal motif (116–120) with ATG4 and RavZ might determine the specificity of cleavage site.     

Chemical Synthesis of Integral Membrane Proteins: Methods and Applications

The development of efficient chemical methods for total synthesis or semisynthesis of integral membrane proteins is an important challenge at the interface between chemistry and biology. This review outlines the recent advances in the synthesis of integral membrane proteins, with particular focus on the methods for difficult peptide synthesis, purification, and enhancement of peptide solubility under the ligation conditions. The applications of these methods to the synthesis of integral membrane proteins with one or multiple transmembrane domains are also described.     

Membrane Fusion and SNAREs: Interaction with Ras Proteins

The superfamily of Ras proteins comprises different molecules belonging to the GTPase family. They normally cycle between an active state bound to GTP which activates effectors while the protein is membrane-associated, and an inactive GDP-bound state. They regulate the intracellular trafficking and other cellular processes. The family of Rab proteins includes several members and they have been found, among other Ras proteins, to be fundamental for important biological processes, such as endocytosis and exocytosis. SNARE proteins control the fusion of vesicles by forming quaternary complexes which are divided into two small groups on the two different compartments. Generally, the association of three SNARE proteins on the donor compartment with the one on the target compartment determines the formation of the SNARE complex, the opening of the fusion pore and the formation of one single bigger vesicle. Interestingly, novel interactions between other molecules involved in intracellular trafficking, endosomal fusion and maturation have recently been found, such as the interaction between invariant chain and the Qb SNARE vti1b, and more functional connections between Rab proteins and SNAREs are supposed to be fundamental for the regulation of membrane fusion.     

Stapled Peptide Inhibitors of Autophagy Adapter LC3B

A growing body of evidence suggests that autophagy inhibition enhances the effectiveness of chemotherapy, especially in difficult-to-treat cancers. Existing autophagy inhibitors are primarily lysosomotropic agents. More specific autophagy inhibitors are highly sought-after. The microtubule-associated protein 1A/1B light chain 3B protein, LC3B, is an adapter protein that mediates key protein-protein interactions at several points in autophagy pathways. In this work, we used a known peptide ligand as a starting point to develop improved LC3B inhibitors. We obtained structure-activity relationships that quantify the binding contributions of peptide termini, individual charged residues, and hydrophobic interactions. Based on these data, we used artificial amino acids and diversity-oriented stapling to improve affinity and resistance to biological degradation, while maintaining or improving LC3B affinity and selectivity. These peptides represent the highest-affinity LC3B-selective ligands reported to date, and they will be useful tools for further elucidation of LC3B's role in autophagy and in cancer.     

Norepinephrine Protects against Methamphetamine Toxicity through β2-Adrenergic Receptors Promoting LC3 Compartmentalization

Norepinephrine (NE) neurons and extracellular NE exert some protective effects against a variety of insults, including methamphetamine (Meth)-induced cell damage. The intimate mechanism of protection remains difficult to be analyzed in vivo. In fact, this may occur directly on target neurons or as the indirect consequence of NE-induced alterations in the activity of trans-synaptic loops. Therefore, to elude neuronal networks, which may contribute to these effects in vivo, the present study investigates whether NE still protects when directly applied to Meth-treated PC12 cells. Meth was selected based on its detrimental effects along various specific brain areas. The study shows that NE directly protects in vitro against Meth-induced cell damage. The present study indicates that such an effect fully depends on the activation of plasma membrane β2-adrenergic receptors (ARs). Evidence indicates that β2-ARs activation restores autophagy, which is impaired by Meth administration. This occurs via restoration of the autophagy flux and, as assessed by ultrastructural morphometry, by preventing the dissipation of microtubule-associated protein 1 light chain 3 (LC3) from autophagy vacuoles to the cytosol, which is produced instead during Meth toxicity. These findings may have an impact in a variety of degenerative conditions characterized by NE deficiency along with autophagy impairment.     

Steroid‐Based Amphiphiles for Membrane Protein Study: The Importance of Alkyl Spacers for Protein Stability

Membrane proteins allow effective communication between cells and organelles and their external environments. Maintaining membrane protein stability in a non‐native environment is the major bottleneck to their structural study. Detergents are widely used to extract membrane proteins from the membrane and to keep the extracted protein in a stable state for downstream characterisation. In this study, three sets of steroid‐based amphiphiles—glyco‐diosgenin analogues (GDNs) and steroid‐based pentasaccharides either lacking a linker (SPSs) or containing a linker (SPS‐Ls)—have been developed as new chemical tools for membrane protein research. These detergents were tested with three membrane proteins in order to characterise their ability to extract membrane proteins from the membrane and to stabilise membrane proteins long‐term. Some of the detergents, particularly the SPS‐Ls, displayed favourable behaviour with the tested membrane proteins. This result indicates the potential utility of these detergents as chemical tools for membrane protein structural study and a critical role of the simple alkyl spacer in determining detergent efficacy.     

Production of an Active,Human Membrane Protein in Saccharomyces cerevisiae: Full-Length FICD

The human Fic domain-containing protein (FICD) is a type II endoplasmic reticulum (ER) membrane protein that is important for the maintenance of ER proteostasis. Structural and in vitro biochemical characterisation of FICD AMPylase and deAMPylase activity have been restricted to the soluble ER-luminal domain produced in Escherichia coli. Information about potentially important features, such as structural motifs, modulator binding sites or other regulatory elements, is therefore missing for the approximately 100 N-terminal residues including the transmembrane region of FICD. Expressing and purifying the required quantity and quality of membrane proteins is demanding because of the low yields and poor stability often observed. Here, we produce full-length FICD by combining a Saccharomyces cerevisiae-based platform with green fluorescent protein (GFP) tagging to optimise the conditions for expression, solubilisation and purification. We subsequently employ these conditions to purify milligram quantities of His-tagged FICD per litre of culture, and show that the purified, detergent-solubilised membrane protein is an active deAMPylating enzyme. Our work provides a straightforward methodology for producing not only full-length FICD, but also other membrane proteins in S. cerevisiae for structural and biochemical characterisation.     

Expanding the Scope of Protein Trans‐Splicing to Fragment Ligation of an Integral Membrane Protein: Towards Modulation of Porin‐Based Ion Channels by Chemical Modification

It's raining, it's porin : Fragment ligation of OmpF ion channels was achieved by using the split Psp‐GBD Pol intein; this allowed reconstitution of active trimeric porin. In combination with cysteine modification at an internal position, the porin's conductance properties were altered.

     

Solution‐NMR Characterization of Outer‐Membrane Protein A from E. coli in Lipid Bilayer Nanodiscs and Detergent Micelles

X‐ray crystallography and solution NMR of detergent‐reconstituted OmpA (outer membrane protein A from E. coli) had shown that this protein forms an eight‐stranded transmembrane β‐barrel, but only limited information was obtained for the extracellular loops. In NMR studies of OmpA in two different detergent micelles, “NMR‐invisible” amino acid residues in‐between the extracellular loops and the β‐barrel prevented complete structural characterization. Here, we show that this NMR‐invisible ring around the β‐barrel of OmpA is also present in lipid bilayer nanodiscs and in mixed micelles with a third detergent, thus suggesting that the implicated rate processes have a functional role rather than representing an artifact of the protein reconstitution. In addition to sequence‐specific NMR assignments for OmpA in the nanodiscs, the present results are based on a protocol of micro‐coil TROSY‐ and CRINEPT‐type NMR diffusion measurements for studying the hydrodynamic properties and the foldedness of [²H,¹⁵N]‐labeled membrane proteins in nanodiscs. This protocol can be applied under conditions closely similar to those used for NMR structure determinations or crystallization trials.     

Scanning Electron-Assisted Dielectric Microscopy Reveals Autophagosome Formation by LC3 and ATG12 in Cultured Mammalian Cells

Autophagy is an intracellular self-devouring system that plays a central role in cellular recycling. The formation of functional autophagosomes depends on several autophagy-related proteins, including the microtubule-associated proteins 1A/1B light chain 3 (LC3) and the conserved autophagy-related gene 12 (Atg12). We have recently developed a novel scanning electron-assisted dielectric microscope (SE-ADM) for nanoscale observations of intact cells. Here, we used the SE-ADM system to observe LC3- and Atg12-containing autophagosomes in cells labelled in the culture medium with antibodies conjugated to colloidal gold particles. We observed that, during autophagosome formation, Atg12 localized along the actin meshwork structure, whereas LC3 formed arcuate or circular alignments. Our system also showed a difference in the distribution of LC3 and Atg12; Atg12 was broadly distributed while LC3 was more localized. The difference in the spatial distribution demonstrated by our system explains the difference in the size of fluorescent spots due to the fluorescently labelled antibodies observed using optical microscopy. The direct SE-ADM observation of cells should thus be effective in analyses of autophagosome formation.     

Isomeric Detergent Comparison for Membrane Protein Stability: Importance of Inter‐Alkyl‐Chain Distance and Alkyl Chain Length

Membrane proteins encapsulated by detergent micelles are widely used for structural study. Because of their amphipathic property, detergents have the ability to maintain protein solubility and stability in an aqueous medium. However, conventional detergents have serious limitations in their scope and utility, particularly for eukaryotic membrane proteins and membrane protein complexes. Thus, a number of new agents have been devised; some have made significant contributions to membrane protein structural studies. However, few detergent design principles are available. In this study, we prepared meta and ortho isomers of the previously reported para‐substituted xylene‐linked maltoside amphiphiles (XMAs), along with alkyl chain‐length variation. The isomeric XMAs were assessed with three membrane proteins, and the meta isomer with a C₁₂ alkyl chain was most effective at maintaining solubility/stability of the membrane proteins. We propose that interplay between the hydrophile–lipophile balance (HLB) and alkyl chain length is of central importance for high detergent efficacy. In addition, differences in inter‐alkyl‐chain distance between the isomers influence the ability of the detergents to stabilise membrane proteins.     

Evolution of the Membrane Transport Protein Domain

Membrane transport proteins are widely present in all living organisms, however, their function, transported substrate, and mechanism of action are unknown. Here we use diverse bioinformatics tools to investigate the evolution of MTPs, analyse domain organisation and loop topology, and study the comparative alignment of modelled 3D structures. Our results suggest a high level of conservancy between MTPs from different taxa on both amino acids and structural levels, which imply some degree of functional similarities. The presence of loop/s of different lengths in various positions suggests tax-on-specific adaptation to transported substrates, intracellular localisation, accessibility for post-translation modifications, and interaction with other proteins. The comparison of modelled structures proposes close relations and a common origin for MTP and Na/H exchanger. Further, a high level of amino acid similarity and identity between archaeal and bacterial MTPs and Na/H exchangers imply conservancy of ion transporting function at least for archaeal and bacterial MTPs.     

草原兔尾鼠卵透明带3融合蛋白抗原的制备及免疫反应性

目的大量制备重组的草原兔尾鼠卵透明带3(Recom binant Laguruslagurus zona pellucida 3,r-LZP3)融合蛋白,并检测其免疫反应性。方法经密码子优化的Lzp3基因在E.coli BL21(DE3)中以包涵体形式表达融合蛋白,表达产物经洗涤、纯化、溶解、复性后,用Western blot、ELISA和抗生育试验鉴定免疫反应性。结果得到纯度达93%的r-LZP3蛋白。Western blot显示表达产物与LZP3小鼠抗血清出现特异性反应条带。ELISA表明免疫小鼠血清中LZP3抗体滴度达1∶45000以上。抗生育试验表明r-LZP3蛋白免疫小鼠的避孕率为25%,平均每胎产仔数减少4~5只。结论已获得了大量具有显著免疫活性的r-LZP3蛋白,为控制草原兔尾鼠鼠害的研究提供了重要的抗原。     

Cell‐Free Protein Synthesis: Pros and Cons of Prokaryotic and Eukaryotic Systems

From its start as a small‐scale in vitro system to study fundamental translation processes, cell‐free protein synthesis quickly rose to become a potent platform for the high‐yield production of proteins. In contrast to classical in vivo protein expression, cell‐free systems do not need time‐consuming cloning steps, and the open nature provides easy manipulation of reaction conditions as well as high‐throughput potential. Especially for the synthesis of difficult to express proteins, such as toxic and transmembrane proteins, cell‐free systems are of enormous interest. The modification of the genetic code to incorporate non‐canonical amino acids into the target protein in particular provides enormous potential in biotechnology and pharmaceutical research and is in the focus of many cell‐free projects. Many sophisticated cell‐free systems for manifold applications have been established. This review describes the recent advances in cell‐free protein synthesis and details the expanding applications in this field.     

Comparison of Membrane Targeting Strategies for the Accumulation of the Human Immunodeficiency Virus p24 Protein in Transgenic Tobacco

Membrane anchorage was tested as a strategy to accumulate recombinant proteins in transgenic plants. Transmembrane domains of different lengths and topology were fused to the cytosolic HIV antigen p24, to promote endoplasmic reticulum (ER) residence or traffic to distal compartments of the secretory pathway in transgenic tobacco. Fusions to a domain of the maize seed storage protein γ-zein were also expressed, as a reference strategy that leads to very high stability via the formation of large polymers in the ER lumen. Although all the membrane anchored constructs were less stable compared to the zein fusions, residence at the ER membrane either as a type I fusion (where the p24 sequence is luminal) or a tail-anchored fusion (where the p24 sequence is cytosolic) resulted in much higher stability than delivery to the plasma membrane or intermediate traffic compartments. Delivery to the tonoplast was never observed. The inclusion of a thrombin cleavage site allowed for the quantitative in vitro recovery of p24 from all constructs. These results point to the ER as suitable compartment for the accumulation of membrane-anchored recombinant proteins in plants.     

Effects of Sulforaphane-Induced Cell Death upon Repeated Passage of Either P-Glycoprotein-Negative or P-Glycoprotein-Positive L1210 Cell Variants

The expression of the membrane ABCB1 transporter in neoplastic cells is one of the most common causes of reduced sensitivity to chemotherapy. In our previous study, we investigated the effect of a single culture of ABCB1-negative (S) and ABCB1-positive variants of L1210 cells (R and T) in the presence of sulforaphane (SFN). We demonstrated that SFN induces the onset of autophagy more markedly in S cells than in R or T cells. In the current study, we focused on the effect of the repeated culture of S, R and T cells in SFN-containing media. The repeated cultures increased the onset of autophagy compared to the simple culture, mainly in S cells and to a lesser extent in R and T cells, as indicated by changes in the cellular content of 16 and 18 kDa fragments of LC3B protein or changes in the specific staining of cells with monodansylcadaverine. We conclude that SFN affects ABCB1-negative S cells more than ABCB1-positive R and T cells during repeated culturing. Changes in cell sensitivity to SFN appear to be related to the expression of genes for cell-cycle checkpoints, such as cyclins and cyclin-dependent kinases.     

荧光磁性双功能Fe3O4@PHEMA-Tb微球的制备及其蛋白固定化

以甲基丙烯酸-2-羟基乙酯为单体,N,N￠-亚甲基双丙烯酰胺为交联剂,采用光化学方法在Fe3O4磁性液体中制备了磁性聚甲基丙烯酸-2-羟基乙酯微球,合成了含有稀土元素Tb的荧光磁性高分子微球,以牛血清白蛋白为模型对微球固定释放蛋白能力进行了研究,用VSM, PCS, FT-IR, TG-DTA, SEM, FS, UV-Vis等技术对微球的性能进行了表征. 结果表明,荧光磁性高分子微球粒径为29.6 nm,比饱和磁化强度为40.1 emu/g,变异系数为3.7%,具有超顺磁性荧光性,分散性好,呈圆球形,对蛋白的装载率和包封率分别为6.5%和74.7%,pH越低蛋白释放率越高.