Editorial : Autophagy : from big data to physiological significance

Autophagy is a fundamental catabolic process where cytoplasmic components are sequestered into double-membrane vesicles called autophagosomes, which then fuse with lysosomes and their content is degraded. Our knowledge about autophagy sharply increased during the last decade. This significant progress helped us to understand better the molecular mechanisms of autophagy and to elucidate its role in health and disease. This special issue contains a collection of three original research papers and 12 review articles covering a broad range of topics highlighting how big data and screening approaches can help toward uncovering the molecular mechanisms of autophagy

iLIR : a web resource for prediction of Atg8-family interacting proteins

Macroautophagy was initially considered to be a nonselective process for bulk breakdown of cytosolic material. However, recent evidence points toward a selective mode of autophagy mediated by the so-called selective autophagy receptors (SARs). SARs act by recognizing and sorting diverse cargo substrates (e.g., proteins, organelles, pathogens) to the autophagic machinery. Known SARs are characterized by a short linear sequence motif (LIR-, LRS-, or AIM-motif) responsible for the interaction between SARs and proteins of the Atg8 family. Interestingly, many LIR-containing proteins (LIRCPs) are also involved in autophagosome formation and maturation and a few of them in regulating signaling pathways. Despite recent research efforts to experimentally identify LIRCPs, only a few dozen of this class of—often unrelated—proteins have been characterized so far using tedious cell biological, biochemical, and crystallographic approaches. The availability of an ever-increasing number of complete eukaryotic genomes provides a grand challenge for characterizing novel LIRCPs throughout the eukaryotes. Along these lines, we developed iLIR, a freely available web resource, which provides in silico tools for assisting the identification of novel LIRCPs. Given an amino acid sequence as input, iLIR searches for instances of short sequences compliant with a refined sensitive regular expression pattern of the extended LIR motif (xLIR-motif) and retrieves characterized protein domains from the SMART database for the query. Additionally, iLIR scores xLIRs against a custom position-specific scoring matrix (PSSM) and identifies potentially disordered subsequences with protein interaction potential overlapping with detected xLIR-motifs. Here we demonstrate that proteins satisfying these criteria make good LIRCP candidates for further experimental verification. Domain architecture is displayed in an informative graphic, and detailed results are also available in tabular form. We anticipate that iLIR will assist with elucidating the full complement of LIRCPs in eukaryotes

Measurement of the Bottom-Strange Meson Mixing Phase in the Full CDF Data Set

We report a measurement of the bottom-strange meson mixing phase \beta_s using the time evolution of B0_s -> J/\psi (->\mu+\mu-) \phi (-> K+ K-) decays in which the quark-flavor content of the bottom-strange meson is identified at production. This measurement uses the full data set of proton-antiproton collisions at sqrt(s)= 1.96 TeV collected by the Collider Detector experiment at the Fermilab Tevatron, corresponding to 9.6 fb-1 of integrated luminosity. We report confidence regions in the two-dimensional space of \beta_s and the B0_s decay-width difference \Delta\Gamma_s, and measure \beta_s in [-\pi/2, -1.51] U [-0.06, 0.30] U [1.26, \pi/2] at the 68% confidence level, in agreement with the standard model expectation. Assuming the standard model value of \beta_s, we also determine \Delta\Gamma_s = 0.068 +- 0.026 (stat) +- 0.009 (syst) ps-1 and the mean B0_s lifetime, \tau_s = 1.528 +- 0.019 (stat) +- 0.009 (syst) ps, which are consistent and competitive with determinations by other experiments.Comment: 8 pages, 2 figures, Phys. Rev. Lett 109, 171802 (2012

Essential versus accessory aspects of cell death: recommendations of the NCCD 2015

Cells exposed to extreme physicochemical or mechanical stimuli die in an uncontrollable manner, as a result of their immediate structural breakdown. Such an unavoidable variant of cellular demise is generally referred to as ‘accidental cell death’ (ACD). In most settings, however, cell death is initiated by a genetically encoded apparatus, correlating with the fact that its course can be altered by pharmacologic or genetic interventions. ‘Regulated cell death’ (RCD) can occur as part of physiologic programs or can be activated once adaptive responses to perturbations of the extracellular or intracellular microenvironment fail. The biochemical phenomena that accompany RCD may be harnessed to classify it into a few subtypes, which often (but not always) exhibit stereotyped morphologic features. Nonetheless, efficiently inhibiting the processes that are commonly thought to cause RCD, such as the activation of executioner caspases in the course of apoptosis, does not exert true cytoprotective effects in the mammalian system, but simply alters the kinetics of cellular demise as it shifts its morphologic and biochemical correlates. Conversely, bona fide cytoprotection can be achieved by inhibiting the transduction of lethal signals in the early phases of the process, when adaptive responses are still operational. Thus, the mechanisms that truly execute RCD may be less understood, less inhibitable and perhaps more homogeneous than previously thought. Here, the Nomenclature Committee on Cell Death formulates a set of recommendations to help scientists and researchers to discriminate between essential and accessory aspects of cell death

Caspase involvement in autophagy

Caspases are a family of cysteine proteases widely known as the principal mediators of the apoptotic cell death response, but considerably less so as the contributors to the regulation of pathways outside cellular demise. In regards to autophagy, the modulatory roles of caspases have only recently begun to be adequately described. In contrast to apoptosis, autophagy promotes cell survival by providing energy and nutrients through the lysosomal degradation of cytoplasmic constituents. Under basal conditions autophagy and apoptosis cross-regulate each other through an elaborate network of interconnections which also includes the interplay between autophagyrelated proteins (ATGs) and caspases. In this review we focus on the effects of this crosstalk at the cellular level, as we aim to concentrate the main observations from research conducted so far on the fine-tuning of autophagy by caspases. Several members of this protease-family have been found to directly interact with key ATGs involved in different tiers across the autophagic cascade. Therefore, we firstly outline the core mechanism of macroautophagy in brief. In an effort to emphasize the importance of the intricate cross-regulation of ATGs and caspases, we also present examples drawn from Drosophila and plant models regarding the contribution of autophagy to apoptotic cell death during normal development

Intermediate filament cytoskeleton of the liver in health and disease

Intermediate filaments (IFs) represent the largest cytoskeletal gene family comprising ~70 genes expressed in tissue specific manner. In addition to scaffolding function, they form complex signaling platforms and interact with various kinases, adaptor, and apoptotic proteins. IFs are established cytoprotectants and IF variants are associated with >30 human diseases. Furthermore, IF-containing inclusion bodies are characteristic features of several neurodegenerative, muscular, and other disorders. Acidic (type I) and basic keratins (type II) build obligatory type I and type II heteropolymers and are expressed in epithelial cells. Adult hepatocytes contain K8 and K18 as their only cytoplasmic IF pair, whereas cholangiocytes express K7 and K19 in addition. K8/K18-deficient animals exhibit a marked susceptibility to various toxic agents and Fas-induced apoptosis. In humans, K8/K18 variants predispose to development of end-stage liver disease and acute liver failure (ALF). K8/K18 variants also associate with development of liver fibrosis in patients with chronic hepatitis C. Mallory-Denk bodies (MDBs) are protein aggregates consisting of ubiquitinated K8/K18, chaperones and sequestosome1/p62 (p62) as their major constituents. MDBs are found in various liver diseases including alcoholic and non-alcoholic steatohepatitis and can be formed in mice by feeding hepatotoxic substances griseofulvin and 3,5-diethoxycarbonyl-1,4-dihydrocollidine (DDC). MDBs also arise in cell culture after transfection with K8/K18, ubiquitin, and p62. Major factors that determine MDB formation in vivo are the type of stress (with oxidative stress as a major player), the extent of stress-induced protein misfolding and resulting chaperone, proteasome and autophagy overload, keratin 8 excess, transglutaminase activation with transamidation of keratin 8 and p62 upregulation

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