The Carboxyl-Terminal Amino Acids Render Pro-Human LC3B Migration Similar to Lipidated LC3B in SDS-PAGE

LC3 is widely used marker for macroautophagy assays. After translation pro-LC3 is processed by Atg4 to expose C-terminal glycine residue for downstream conjugation reactions to accomplish the conversion of LC3-I to LC3-II. SDS-PAGE based Western blot (Wb) is generally utilized to quantify LC3-II levels where the LC3-I band migrates slower than LC3-II. We found that pro-human LC3B migrated at similar rate as LC3B-II in SDS-PAGE. The carboxyl-terminal five amino acids, particularly Lysine122 and Leucine123 of human LC3B play a major role in the faster migration of unprocessed LC3B, rendering it indistinguishable from LC3B-II in Wb assays. The unique faster migration of unprocessed LC3B than LC3B-I is also revealed in mouse LC3B, rat LC3B and rat LC3 but not in human LC3C. Our findings for the first time define pro-LC3 migration patterns for LC3 family member from human, mouse and rat species in SDS-PAGE. These findings provide a reference for pro-LC3 band patterns when Atg4 function is inhibited. © 2013 Wang et al

E1-Like Activating Enzyme Atg7 Is Preferentially Sequestered into p62 Aggregates via Its Interaction with LC3-I

p62 is constitutively degraded by autophagy via its interaction with LC3. However, the interaction of p62 with LC3 species in the context of the LC3 lipidation process is not specified. Further, the p62-mediated protein aggregation's effect on autophagy is unclear. We systemically analyzed the interactions of p62 with all known Atg proteins involved in LC3 lipidation. We find that p62 does not interact with LC3 at the stages when it is being processed by Atg4B or when it is complexed or conjugated with Atg3. p62 does interact with LC3-I and LC3-I:Atg7 complex and is preferentially recruited by LC3-II species under autophagic stimulation. Given that Atg4B, Atg3 and LC3-Atg3 are indispensable for LC3-II conversion, our study reveals a protective mechanism for Atg4B, Atg3 and LC3-Atg3 conjugate from being inappropriately sequestered into p62 aggregates. Our findings imply that p62 could potentially impair autophagy by negatively affecting LC3 lipidation and contribute to the development of protein aggregate diseases. © 2013 Gao et al

A protein functionalization platform based on selective reactions at methionine residues.

Nature has a remarkable ability to carry out site-selective post-translational modification of proteins, therefore enabling a marked increase in their functional diversity1. Inspired by this, chemical tools have been developed for the synthetic manipulation of protein structure and function, and have become essential to the continued advancement of chemical biology, molecular biology and medicine. However, the number of chemical transformations that are suitable for effective protein functionalization is limited, because the stringent demands inherent to biological systems preclude the applicability of many potential processes2. These chemical transformations often need to be selective at a single site on a protein, proceed with very fast reaction rates, operate under biologically ambient conditions and should provide homogeneous products with near-perfect conversion2-7. Although many bioconjugation methods exist at cysteine, lysine and tyrosine, a method targeting a less-explored amino acid would considerably expand the protein functionalization toolbox. Here we report the development of a multifaceted approach to protein functionalization based on chemoselective labelling at methionine residues. By exploiting the electrophilic reactivity of a bespoke hypervalent iodine reagent, the S-Me group in the side chain of methionine can be targeted. The bioconjugation reaction is fast, selective, operates at low-micromolar concentrations and is complementary to existing bioconjugation strategies. Moreover, it produces a protein conjugate that is itself a high-energy intermediate with reactive properties and can serve as a platform for the development of secondary, visible-light-mediated bioorthogonal protein functionalization processes. The merger of these approaches provides a versatile platform for the development of distinct transformations that deliver information-rich protein conjugates directly from the native biomacromolecules

Targeting Antibody Responses to the Membrane Proximal External Region of the Envelope Glycoprotein of Human Immunodeficiency Virus

Although human immunodeficiency type 1 (HIV-1) infection induces strong antibody responses to the viral envelope glycoprotein (Env) only a few of these antibodies possess the capacity to neutralize a broad range of strains. The induction of such antibodies represents an important goal in the development of a preventive vaccine against the infection. Among the broadly neutralizing monoclonal antibodies discovered so far, three (2F5, Z13 and 4E10) target the short and hidden membrane proximal external region (MPER) of the gp41 transmembrane protein. Antibody responses to MPER are rarely observed in HIV-infected individuals or after immunization with Env immunogens. To initiate antibody responses to MPER in its membrane-embedded native conformation, we generated expression plasmids encoding the membrane-anchored ectodomain of gp41 with N-terminal deletions of various sizes. Following transfection of these plasmids, the MPER domains are displayed on the cell surface and incorporated into HIV virus like particles (VLP). Transfected cells displaying MPER mutants bound as efficiently to both 2F5 and 4E10 as cells transfected with a plasmid encoding full-length Env. Mice immunized with VLPs containing the MPER mutants produced MPER-specific antibodies, the levels of which could be increased by the trimerization of the displayed proteins as well as by a DNA prime-VLP boost immunization strategy. Although 2F5 competed for binding to MPER with antibodies in sera of some of the immunized mice, neutralizing activity could not be detected. Whether this is due to inefficient binding of the induced antibodies to MPER in the context of wild type Env or whether the overall MPER-specific antibody response induced by the MPER display mutants is too low to reveal neutralizing activity, remains to be determined

Mechanical Bonds and Topological Effects in Radical Dimer Stabilization

While mechanical bonding stabilizes tetrathiafulvalene (TTF) radical dimers, the question arises: what role does topology play in catenanes containing TTF units? Here, we report how topology, together with mechanical bonding, in isomeric [3]- and doubly interlocked [2]catenanes controls the formation of TTF radical dimers within their structural frameworks, including a ring-in-ring complex (formed between an organoplatinum square and a {2+2} macrocyclic polyether containing two 1,5-dioxynaphthalene (DNP) and two TTF units) that is topologically isomeric with the doubly interlocked [2]catenane. The separate TTF units in the two {1+1} macrocycles (each containing also one DNP unit) of the isomeric [3]catenane exhibit slightly different redox properties compared with those in the {2+2} macrocycle present in the [2]catenane, while comparison with its topological isomer reveals substantially different redox behavior. Although the stabilities of the mixed-valence (TTF2)^(•+) dimers are similar in the two catenanes, the radical cationic (TTF^(•+))_2 dimer in the [2]catenane occurs only fleetingly compared with its prominent existence in the [3]catenane, while both dimers are absent altogether in the ring-in-ring complex. The electrochemical behavior of these three radically configurable isomers demonstrates that a fundamental relationship exists between topology and redox properties

Visualization and Movement as Configurations of Human-Nonhuman Engagements : Precolonial Geometric Earthwork Landscapes of the Upper Purus, Brazil

Producing geometric designs and images on materials, such as pottery, basketry, and bead artwork, as well as the human body, is elemental and widespread among Amazonian Indigenous peoples. In this article, we examine the different geometric forms identified in the precolonial geoglyph architecture of southwestern Amazonia in the context of geometric design making and relational ontologies. Our aim is to explore earthwork iconography through the lens of Amerindian visual arts and movement. Combining ethnographic and archaeological data from the Upper Purus, Brazil, the article shows how ancient history and socio-cosmology are deeply "written" onto the landscape in the form of geometric earthworks carved out of the soil, which materialize interactions between nonhuman and human actors. We underline skills in visualization, imaginative practices, and movement as ways to promote well-balanced engagements with animated life forms. Here, iconography inserted in the landscape is both a form of writing and also emerges as an agent, affecting people through visual and corporal practices.Peer reviewe

Guidelines for the use and interpretation of assays for monitoring autophagy (3rd edition)

In 2008 we published the first set of guidelines for standardizing research in autophagy. Since then, research on this topic has continued to accelerate, and many new scientists have entered the field. Our knowledge base and relevant new technologies have also been expanding. Accordingly, it is important to update these guidelines for monitoring autophagy in different organisms. Various reviews have described the range of assays that have been used for this purpose. Nevertheless, there continues to be confusion regarding acceptable methods to measure autophagy, especially in multicellular eukaryotes. For example, a key point that needs to be emphasized is that there is a difference between measurements that monitor the numbers or volume of autophagic elements (e.g., autophagosomes or autolysosomes) at any stage of the autophagic process versus those that measure fl ux through the autophagy pathway (i.e., the complete process including the amount and rate of cargo sequestered and degraded). In particular, a block in macroautophagy that results in autophagosome accumulation must be differentiated from stimuli that increase autophagic activity, defi ned as increased autophagy induction coupled with increased delivery to, and degradation within, lysosomes (inmost higher eukaryotes and some protists such as Dictyostelium ) or the vacuole (in plants and fungi). In other words, it is especially important that investigators new to the fi eld understand that the appearance of more autophagosomes does not necessarily equate with more autophagy. In fact, in many cases, autophagosomes accumulate because of a block in trafficking to lysosomes without a concomitant change in autophagosome biogenesis, whereas an increase in autolysosomes may reflect a reduction in degradative activity. It is worth emphasizing here that lysosomal digestion is a stage of autophagy and evaluating its competence is a crucial part of the evaluation of autophagic flux, or complete autophagy. Here, we present a set of guidelines for the selection and interpretation of methods for use by investigators who aim to examine macroautophagy and related processes, as well as for reviewers who need to provide realistic and reasonable critiques of papers that are focused on these processes. These guidelines are not meant to be a formulaic set of rules, because the appropriate assays depend in part on the question being asked and the system being used. In addition, we emphasize that no individual assay is guaranteed to be the most appropriate one in every situation, and we strongly recommend the use of multiple assays to monitor autophagy. Along these lines, because of the potential for pleiotropic effects due to blocking autophagy through genetic manipulation it is imperative to delete or knock down more than one autophagy-related gene. In addition, some individual Atg proteins, or groups of proteins, are involved in other cellular pathways so not all Atg proteins can be used as a specific marker for an autophagic process. In these guidelines, we consider these various methods of assessing autophagy and what information can, or cannot, be obtained from them. Finally, by discussing the merits and limits of particular autophagy assays, we hope to encourage technical innovation in the field