hfAIM: A reliable bioinformatics approach for in silico genome-wide identification of autophagy-associated Atg8-interacting motifs in various organisms

Most of the proteins that are specifically turned over by selective autophagy are recognized by the presence of short Atg8 interacting motifs (AIMs) that facilitate their association with the autophagy apparatus. Such AIMs can be identified by bioinformatics methods based on their defined degenerate consensus F/W/Y-X-X-L/I/V sequences in which X represents any amino acid. Achieving reliability and/or fidelity of the prediction of such AIMs on a genome-wide scale represents a major challenge. Here, we present a bioinformatics approach, high fidelity AIM (hfAIM), which uses additional sequence requirements—the presence of acidic amino acids and the absence of positively charged amino acids in certain positions—to reliably identify AIMs in proteins. We demonstrate that the use of the hfAIM method allows for in silico high fidelity prediction of AIMs in AIM-containing proteins (ACPs) on a genome-wide scale in various organisms. Furthermore, by using hfAIM to identify putative AIMs in the Arabidopsis proteome, we illustrate a potential contribution of selective autophagy to various biological processes. More specifically, we identified 9 peroxisomal PEX proteins that contain hfAIM motifs, among which AtPEX1, AtPEX6 and AtPEX10 possess evolutionary-conserved AIMs. Bimolecular fluorescence complementation (BiFC) results verified that AtPEX6 and AtPEX10 indeed interact with Atg8 in planta. In addition, we show that mutations occurring within or nearby hfAIMs in PEX1, PEX6 and PEX10 caused defects in the growth and development of various organisms. Taken together, the above results suggest that the hfAIM tool can be used to effectively perform genome-wide in silico screens of proteins that are potentially regulated by selective autophagy. The hfAIM system is a web tool that can be accessed at link: http://bioinformatics.psb.ugent.be/hfAIM/.     

Fate of adenovirus type 12 genomes in nonpermissive cells

The fate of ³H-thymidine-labeled adenovirus type 12 deoxyribonucleic acid (DNA) was studied in Nil-2 cells of Syrian hamster origin. It was found that a substantial fraction of ³H-adenovirus type 12 DNA became degraded within 24 hr after infection and was released into the culture fluid. After infection of 5-bromodeoxyuridine (BUdR)-prelabeled cells with ³H-adenovirus type 12, viral DNA became readily separable from cellular DNA by equilibrium centrifugation in CsCl. Part of the viral radioactivity was found to shift gradually to the position of cellular DNA as time progressed after infection. When exponentially growing cells were exposed simultaneously to BUdR, 5-fluorodeoxyuridine, and ³H-adenovirus type 12, up to 50% of the viral radioactivity shifted within 24 hr from the density of viral DNA to that of cellular DNA after equilibrium centrifugation in CsCl. Upon denaturation of the cellular DNA, the isotope was preferentially found to be associated with the “heavy” strand which was synthesized after infection. Upon hybridization of the “heavy” and the “light” strands with sonically treated, denatured ³H-adenovirus type 12 DNA, small and nearly equal amounts of counts hybridized with both strands. The number of counts annealed was in a range similar to that of those annealed with the same amount of DNA derived from adenovirus type 12-transformed hamster cells. These results demonstrate that (i) a substantial proportion of the adsorbed virus becomes degraded within 24 hr; (ii) part of the degradation products is reutilized for cellular DNA synthesis; (iii) only a small fraction, mainly fragments, of viral DNA becomes integrated into both the newly synthesized and the parental strands of cellular DNA.     

All-trans retinoic acid modifies the expression of clock and disease marker genes

Restricted feeding (RF), a regimen that restricts the duration of food availability with no calorie restriction, entrains the circadian clock in peripheral tissues. Restricted feeding leads to high-amplitude circadian rhythms, which have been shown to promote wellness and reduce disease and inflammatory markers. Retinoids, such as all-trans retinoic acid (ATRA), act as anti-inflammatory agents. Thus far, the effect of ATRA combined with RF on the ability to delay the occurrence of age-associated changes, such as cancer and inflammation, is not known. We measured circadian expression of clock genes, disease marker genes and inflammatory markers in the serum, liver and jejunum in mice fed ad libitum (AL) or RF supplemented with 15 or 250 μg/kg body/day ATRA for 16 weeks. Our results show that ATRA supplementation led to phase shifts and reduced amplitudes in clock genes. Under AL, ATRA reduced the average daily messenger RNA (mRNA) levels of some disease markers, such as liver Afp and jejunum Afp, Alt and Gadd45β and aspartate transaminase (AST) protein in the serum, but increased the expression level of liver Crp mRNA. Under RF, ATRA reduced the average daily levels of jejunum Alt and Gadd45β and AST protein in the serum, but increased liver Afp, Alt, Gadd45β and Arginase mRNA. Altogether, our findings suggest that ATRA strongly affects circadian oscillation and disease marker levels. Moreover, its impact is different depending on the feeding regimen (AL or RF).     

Degradation of Arabidopsis CRY2 is regulated by SPA proteins and phytochrome A

The UV-A/blue light photoreceptor crytochrome2 (cry2) plays a fundamental role in the transition from the vegetative to the reproductive phase in the facultative long-day plant Arabidopsis thaliana. The cry2 protein level strongly decreases when etiolated seedlings are exposed to blue light; cry2 is first phosphorylated, polyubiquitinated, and then degraded by the 26S proteasome. COP1 is involved in cry2 degradation, but several cop1 mutants show only reduced but not abolished cry2 degradation. SUPPRESSOR OF PHYA-105 (SPA) proteins are known to work in concert with COP1, and recently direct physical interaction between cry2 and SPA1 was demonstrated. Thus, we hypothesized that SPA proteins could also play a role in cry2 degradation. To this end, we analyzed cry2 protein levels in spa mutants. In all spa mutants analyzed, cry2 degradation under continuous blue light was alleviated in a fluence rate-dependent manner. Consistent with a role of SPA proteins in phytochrome A (phyA) signaling, a phyA mutant had enhanced cry2 levels, particularly under low fluence rate blue light. Fluorescence resonance energy transfer-fluorescence lifetime imaging microscopy studies showed a robust physical interaction of cry2 with SPA1 in nuclei of living cells. Our results suggest that cry2 stability is controlled by SPA and phyA, thus providing more information on the molecular mechanisms of interaction between cryptochrome and phytochrome photoreceptors.     

Aggregation and species coexistence in fleas parasitic on small mammals

The aggregation model of coexistence states that species coexistence is facilitated if interspecific aggregation is reduced relative to intraspecific aggregation. We investigated the relationship between intraspecific and interspecific aggregation in 17 component communities (the flea assemblage of a host population) of fleas parasitic on small mammals and hypothesized that interspecific interactions should be reduced relative to intraspecific interactions, facilitating species coexistence. We predicted that the reduction of the level of interspecific aggregation in relation to the level of intraspecific aggregation would be positively correlated with total flea abundance and species richness of flea assemblages. We also expected that the higher degree of facilitation of flea coexistence would be affected by host parameters such as body mass, basal metabolic rate (BMR) and depth and complexity of burrows. Results of this study supported the aggregation model of coexistence and demonstrated that, in general, a) conspecific fleas were aggregated across their hosts; b) flea assemblages were not dominated by negative interspecific interactions; and c) the level of interspecific aggregation in flea assemblages was reduced in relation to the level of intraspecific aggregation. Intraspecific aggregation tended to be correlated positively to body mass, burrow complexity and mass-independent BMR of a host. Positive interspecific associations of fleas tended to occur more frequently in species-rich flea assemblages and/or in larger hosts possessing deep complex burrows. Intraspecific aggregation increased relative to interspecific aggregation when species richness of flea infracommunities (the flea assemblage of a host individual) and component communities increased. We conclude that the pattern of flea coexistence is related both to the structure of flea communities and affinities of host species.     

Mesoderm progenitor cells of common origin contribute to the head musculature and the cardiac outflow tract

During early embryogenesis, heart and skeletal muscle progenitor cells are thought to derive from distinct regions of the mesoderm (i.e. the lateral plate mesoderm and paraxial mesoderm, respectively). In the present study, we have employed both in vitro and in vivo experimental systems in the avian embryo to explore how mesoderm progenitors in the head differentiate into both heart and skeletal muscles. Using fate-mapping studies, gene expression analyses, and manipulation of signaling pathways in the chick embryo, we demonstrate that cells from the cranial paraxial mesoderm contribute to both myocardial and endocardial cell populations within the cardiac outflow tract. We further show that Bmp signaling affects the specification of mesoderm cells in the head: application of Bmp4, both in vitro and in vivo, induces cardiac differentiation in the cranial paraxial mesoderm and blocks the differentiation of skeletal muscle precursors in these cells. Our results demonstrate that cells within the cranial paraxial mesoderm play a vital role in cardiogenesis, as a new source of cardiac progenitors that populate the cardiac outflow tract in vivo. A deeper understanding of mesodermal lineage specification in the vertebrate head is expected to provide insights into the normal, as well as pathological, aspects of heart and craniofacial development.     

Inhibition of Pseudomonas aeruginosa quorum sensing by AI-2 analogs

Autoinducer-2 (AI-2) has been suggested to serve as a universal interspecies quorum sensing signaling molecule. We have synthesized a set of AI-2 analogs with small incremental changes in alkyl substitution on C-2 and evaluated them for their agonistic and antagonistic potential as quorum sensing (QS) attenuators in two different bacterial species: Pseudomonas aeruginosa and Vibrio harveyi. Unexpectedly, several of the analogs were found to function as synergistic QS agonists in V. harveyi, while two of these analogs inhibit QS in P. aeruginosa.